Process for the determination of peptides corresponding to immunologically important epitopes and their use in a process for determination of antibodies or biotinylated peptides corresponding to immunologically important epitopes, a process for preparing them and compositions containing them

ABSTRACT

The technical problem underlying the present invention is to provide peptides corresponding to immunologically important epitopes on bacterial and viral proteins, as well as the use of said peptides in diagnostic or immunogenic compositions. The invention relates to a process for the in vitro determination of antibodies, wherein the peptides used are biotinylated, particularly in the form of complexes of streptavidin-biotinylated peptides or of avidin-biotinylated peptides.

This is a divisional of application Ser. No. 09/576,824, filed May 23,2000, which is a divisional of application Ser. No. 08/723,425, filedSep. 30, 1996, now U.S. Pat. No. 6,165,730 which is a divisional ofapplication Ser. No. 08/146,028, filed Nov. 22, 1993, now U.S. Pat. No.5,891,640 which is a 371 application of PCT/EP93/00517, filed Mar. 8,1993 which claims benefit of EP 92400598.6, filed Mar. 6, 1992 theentire content of which is hereby incorporated by reference in thisapplication.

The technical problem underlying the present invention is to providepeptides corresponding to immunologically important epitopes onbacterial and viral proteins, as well as the use of said peptides indiagnostic or immunogenic compositions.

Recent developments in genetic engineering as well as the chemistry ofsolid phase peptide synthesis have led to the increasingly wider use ofsynthetic peptides in biochemistry and immunology. Protein sequenceswhich become available as a result of molecular cloning techniques canbe synthesized chemically in large quantities for structural,functional, and immunological studies. Peptides corresponding toimmunologically important epitopes found on viral and bacterial proteinshave also proven to be highly specific reagents which can be used forantibody detection and the diagnosis of infection.

Despite the many advantages synthetic peptides offer, there are a numberof disadvantages associated with their use. Because of their relativelyshort size (generally less than 50 amino acids in length), theirstructures may fluctuate between many different conformations in theabsence of the stabilizing influence of intramolecular interactionspresent in the full-length protein. Furthermore, the small size of thesepeptides means that their chemical properties and solubilities willfrequently be quite different from those of the full-length protein andthat the contribution of individual amino acids in the peptide sequencetoward determining the overall chemical properties of the peptide willbe proportionally greater.

Many immunological assays require that the antigen used for antibodydetection be immobilized on a solid support. Most enzyme-linkedimmunosorbent assays (ELISA) make use of polystyrene as the solid phase.Many proteins can be stably adsorbed to the solid phase and presentsequences which are accessible for subsequent interactions withantibodies. Because of their small size, direct adsorption of peptidesto the solid phase frequently gives rise to unsatisfactory results forany of a number of reasons.

Firstly, the peptide may not possess the correct overall charge or aminoacid composition which would enable the peptide to bind to the solidphase. Secondly, the same amino acid residues which are required forbinding to the solid phase may also be required for antibody recognitionand therefore not available for antibody binding. Thirdly, the peptidemay become fixed in an unfavorable conformation upon binding to thesolid phase which renders it unrecognizable to antibody molecules. Inmany cases, it is neither possible nor necessary to distinguish betweenthese possibilities. Binding to the solid phase can be increased andmade less sensitive to the specific chemical properties of a peptide byfirst coupling the peptide to a large carrier molecule. Typically, thecarrier molecule is a protein.

While the amount of peptide bound to the solid phase, albeit indirectly,can in some cases be increased by this method, this approach suffersfrom the fact that the linkage between the peptide and the carrierprotein frequently involves the side chains of internal trifunctionalamino acids whose integrity may be indispensable for recognition byantibodies. The binding avidity of antisera for the internally modifiedpeptide is frequently very much reduced relative to the unmodifiedpeptide or the native protein.

The production of antisera to synthetic peptide also requires in mostcases that the peptide be coupled to a carrier. Again, the couplingreaction between an internal trifunctional amino acid of the peptide andthe carrier is likely to alter the immunogenic properties of thepeptide.

There exist many methods for performing coupling reactions and most ofthe procedures in current use are discussed in detail in VanRegenmortel, M. H. V., Briand, J. P., Muller, S., and Plaue, S.;Laboratory Techniques in Biochemistry and Molecular Biology, vol. 19,Synthetic Polypeptides as Antigens, Elsevier Press, Amsterdam, New York,Oxford, 1988. In addition to these procedures, unprotected peptides canalso be biotinylated using commercially available reagents such asN-hydroxysuccinimidobiotin or biotinamidocaproate N-hydroxysuccinimideester. Many of these reagents are discussed in Billingsley, M. L.,Pennypacker, K. R., Hoover, C. G., and Kincaid, R. L., Biotechniques(1987)5(1):22-31. Biotinylated peptides are capable of being bound bythe proteins streptavidin and avidin, two proteins which exhibitextraordinarily high affinity binding to biotin.

In certain instances, it is possible to selectively couple biotin to anunprotected peptide or an unprotected peptide to a carrier. This may beaccomplished by synthesizing the peptide with an additionaltrifunctional amino acid added to one of the ends which is capable ofparticipating in the coupling reaction. This approach will only besuccessful, however, as long as this amino acid is not a criticalresidue in the immunogenic sequence of interest and as long as thecoupling agent chosen is sufficiently selective. No single technique isapplicable to all unprotected peptide regardless of their amino acidcomposition.

The etiological agent responsible for non-A, non-B hepatitis has beenidentified and termed hepatitis C virus (HCV). Patent application EP-A-0318 216 discloses sequences corresponding to approximately 80% of theviral genome. The availability of these sequences rapidly led to theelucidation of the remainder of the coding sequences, particularly thoselocated in the 5′ end of the genome (Okamoto; J. Exp. Med. 60, 167-177,1990). The HCV genome is a linear, positive-stranded RNA molecule with alength of approximately 9400 nucleotides. With the exception of rathershort untranslated regions at the termini, the genome consists of onelarge, uninterrupted, open reading frame encoding a polyprotein ofapproximately 3000 amino acids. This polyprotein has been shown to becleaved co-translationally into individual viral structural andnon-structural (NS) regions. The structural protein region is furtherdivided into capsi (Core) and envelope (E1 and E2) proteins. The NSregions are divided into NS-1 and NS-5 regions.

A number of independent patent applications have employed a variety ofstrategies to determine the locations of diagnostically important aminoacid sequences and many regions of the HCV polyprotein.

The NS4 region has mainly been studied in EP-A-0 318 216, EP-A-0 442394, U.S. Pat. No. 5,106,726, EP-A-0 489 986, EP-A-0 484 787, and EP-A-0445 801. Unfortunately only 70% of HCV-infected individuals produceantibodies to NS4, neither the synthetic nor recombinant proteinscontaining sequences from this region are adequate for identifying allinfected serum samples. The nucleocapsid or Core region has been studiedin patent applications EP-A-0 442 394, U.S. Pat. No. 5,106,726, EP-A-0489 986, EP-A-0 445 801, EP-A-0 451 891 and EP-A-0 479 376. It was foundthat these peptides often used as mixtures, were more frequentlyrecognized by antibodies (85-90%) in sera from chronically infectedindividuals than were the peptides derived from NS4. The NS5 region wasstudied in patent applications EP-A-0 489 986 and EP-A-0 468 527.Depending on the serum panel used, more than 60% of NANB hepatitis canbe shown to contain antibodies directed against these peptides. The NS3region was also studied in patent application EP-A-0 468 527. Allavailable evidence suggests that the most dominant epitope of NS3 arediscontinuous in nature and cannot be adequately represented bysynthetic peptides. The E1 region which is potentially interesting as aregion from the outer surface of the virus particles (possibleimmunogenic epitopes) was studied in both patent applications EP-A-0 468527 and EP-A-0 507 615. The E2/NS1 region was studied for the samereason as E1. Comparisons of this region from different HCV variantselucidated that this protein contains variable region which arereminiscent of the HIV V3 loop region of gp120 envelope protein. Fourpeptides were found in EP-A-0 468 527 which were shown to containrelatively infrequently recognized epitopes. Finally, the NS2 region ofHCV was analyzed in EP-A-0 486 527. However, the diagnostic value ofthis region is not clear yet. Virtually all patent applicationsconcerning diagnostically useful synthetic peptides for antibodydetection describe preferred combinations of peptides. Most of theseinclude peptides from the HCV core protein and NS4. In some cases,peptides from NS5 (EP-A-0 489 968 and EP-A-0 468 527), and E1 and E2/NS1are included (EP-A-0 507 615 and EO-A-0 468 527).

Different patent applications have addressed the problem of findingdiagnostically useful epitopes of human immunodeficiency virus (HIV). Animportant immunodominant region containing cyclic HIV-1 and HIV-2peptides was found in patent application EP-A-0 326 490. In EP-A-0 379949, this region was asserted to be even more reactive with HIV-specificantibodies in case a biotin molecule was coupled to these cyclic HIVpeptides. SU-A-161 22 64 also describes the use of a biotinylatedpeptide in a solid phase immunoassay for the detection of HIVantibodies.

Other applications have looked for useful HIV epitopes in thehypervariable V3 loop region of gp120 (such as EP-A-0 448 095 and EP-A-0438 332).

U.S. Pat. No. 4,833,071 provides peptide compositions for detection ofHTLV I antibodies.

Deciding whether or not an epitope is diagnostically useful is notalways straightforward and depends to an extent on the specificconfiguration of the test into which it is incorporated. It should beideally an immunodominant epitope which is recognized by a largepercentage of true positive sera or should be able to complement otherantigens in the test to increase the detection rate. Epitopes which arenot frequently recognized may or may not be diagnostically usefuldepending on the contribution they make towards increasing the detectionrate of antibodies in true positive sera and the extent to whichincorporation of these epitopes has an adverse effect on the sensitivityof the test due to dilution of other stronger epitopes.

Peptides can thus be used to identify regions of proteins which arespecifically recognized by antibodies produced as a result of infectionor immunization. In general, there are two strategies which can befollowed. One of these strategies has been described by Geysen, H. M.,Meloen, R. H., and Bateling, S. J.; Proc. Natl. Acad. Sci. USA (1984)81:3998-4002. This approach involves the synthesis of a large series ofshort, overlapping peptides on polyethylene rods derivatized with anoncleavable linker such that the entire length of the protein orprotein fragment of interest is represented.

The rods are incubated with antisera and antibody binding is detectedusing an anti-immunoglobulin: enzyme conjugate. A positive reactionimmediately identifies the location and sequence of epitopes present inthe protein sequence. This technique has the advantage that all peptidesare uniformly linked to the solid support through theircarboxy-terminus. While this method allows for very accurate mapping oflinear epitopes, the length of the peptides which can be reliablysynthesized on the rods is limited. This may sometimes present problemsif the length of the epitope exceeds the length of the peptidessynthesized.

A second approach to epitope mapping involves the synthesis of largerpeptides, generally between fifteen and thirty amino acids in length,along the sequence of the protein to be analyzed. Consecutive peptidesmay be contiguous but are preferably overlapping. Following cleavage,the evaluation of antibody binding to the individual peptides isassessed and the approximate positions of the epitopes can beidentified. An example of this approach is given in Neurath, A. R.,Strick, N., and Lee, E. S. Y.; J. Gen. Virol. (1990) 71:85-95. Thisapproach has the advantage that longer peptides can be synthesized whichpresumably more closely resemble the homologous sequence in the nativeprotein and which offer better targets for antibody binding. Thedisadvantage of this approach is that each peptide is chemically uniqueand that the conditions under which each peptide can be optimally coatedonto a solid phase for immunological evaluation may vary widely in termsof such factors as pH, ionic strength, and buffer composition. Thequantity of peptide which can be adsorbed onto the solid phase is alsoan uncontrolled factor which is unique for each peptide.

The main purpose of the present invention is to provide modifiedpeptides corresponding to immunologically useful epitopes with saidmodified peptides having superior immunological properties overnon-modified versions of these peptides.

Another aim of the present invention is to provide modified peptidescorresponding to immunologically useful epitopes which could not beidentified through classical epitopes mapping techniques.

Another aim of the present invention is to provide a process for the invitro determination of antibodies using said peptides, with said processbeing easy to perform and amenable to standardization.

Another aim of the invention is to provide a process for thedetermination of peptides corresponding to immunologically importantepitopes on bacterial and viral proteins.

Another aim of the invention is to provide a method for preparingprotein sequences used in any of said methods.

Another aim of the invention is to provide a method for preparingprotein sequence which can be used in a process for the determination oftheir epitopes or in an in vitro method for the determination ofantibodies.

Another aim of the invention is to provide intermediary compounds usefulfor the preparation of peptides used in the above-mentioned methods.

Another aim of the present invention is also to provide compositionscontaining peptides determined to correspond to immunologicallyimportant epitopes on proteins for diagnostic purposes.

Another aim of the present invention is also to provide compositionscontaining peptides determined to correspond to immunologicallyimportant epitopes on proteins for vaccine purposes.

According to the present invention, a series of biotinylated peptidesrepresenting immunologically important regions of viral proteins havebeen identified and prepared by solid phase peptide synthesis. Thesepeptides have been identified to be very useful for (i) the detection ofantibodies to HCV, and/or HIV, and/or HTLV-I or II. In some preferredarrangements, these peptides were also found or are at least expected,to be useful in stimulating the production of antibodies to HCV, and/orHIV, and/or HTLV-I or II in healthy animals such as BALB/C mice, and ina vaccine composition to prevent HCV and/or HIV, and/or HTLV-I or IIinfection.

As demonstrated in the Examples section of the present invention, theuse of biotinylated peptides also allows the determination ofimmunologically important epitopes within a previously determinedprotein sequence. The determination of immunologically importantepitopes using non-biotinylated peptides, which are covalently coupledto the solid phase, often fails to localize these epitopes. Especiallyin case of localization of structural epitopes, the use of biotinylatedpeptides seems to be quite successful.

(1) According to the present invention, a peptide composition useful forthe detection of antibodies to HCV, and/or HIV, and/or HTLV-I or IIcomprise peptides corresponding to immunologically important epitopesbeing of the structure:

(A)-(B)-(X)-Y-[amino acids]_(n)-Y-(X)-Z

where

[amino acids]_(n) is meant to designate the length of the peptide chainwhere n is the number of residues, being an integer from about 4 toabout 50, preferably less than about 35, more preferably less than about30, and advantageously from about 4 to about 25;

B represents biotin;

X represents a biotinylated compound which is incorporated during thesynthetic process;

Y represents a covalent bond or one or more chemical entities whichsingly or together form a linker arm separating the amino acids of thepeptide proper from the biotinyl moiety B or X, the function of which isto minimize steric hindrance which may interfere with the binding of thebiotinyl moiety B or X to avidin or streptavidin, wherein Y is not acovalent bond, it is advantageously at least one chemical entity and mayconsist of as many as 30 chemical entities but will consist mostfrequently of 1 to 10 chemical entities, which may be identical ordifferent, more preferably glycine residues, β-alanine, 4-aminobutyricacid, 5-aminovaleric acid, or 6-aminohexanoic acid;

B and X being enclosed in parentheses to indicate that the presence ofbiotin or a biotinylated compound in these positions is optional, theonly stipulation being that B or X be present in at least one of thepositions shown;

A, when present, as indicated by parentheses, represents (an) aminoacid(s), an amino group, or a chemical modification of the aminoterminus of the peptide chain;

Z represents (an) amino acid(s), an OH-group, an NH2-group, or a linkageinvolving either of these two chemical groups wherein the amino acidsare selectively chosen to be immunodominant epitopes which arerecognized by a large percentage of true positive sera or are able tocomplement other antigens in that the test to increase the detectionrate and B interacts with the selected amino acids to produce a compoundwith greater diagnostic sensitivity.

The peptide composition comprises at least one and preferably acombination of two, three, four or more biotinylated peptides chosenfrom the following sequences:

1. Human immunodeficiency Virus type 1 Envelope Peptides:

a. gp41

1. gp41, isolate HTLV-IIIB(A)-(B)-(X)-Y-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Ile-Cys-Y-(X)-Z

2.(A)-(B)-X)-Y-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-Val-Pro-Trp-Asn-Ala-Ser-Y-(X)-Z

3.(A)-(B)-(X)-Y-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Y-(X)-Z

4.(A)-(B)-(X)-Y-Leu-Gln-Ala-Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Leu-Y-(X)-Z

5. gp41, isolate Ant70(A)-(B)-(X)-Y-Leu-Trp-Gly-Cys-Lys-Gly-Lys-Leu-Val-Cys-Y-(X)-Z

6. gp41, isolate ELI(A)-(B)-(X)-Y-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-His-Ile-Cys-Thr-Thr-Asn-Val-Pro-Trp-Asn-Y-(X)-Z

b. gp 120

1. Partial V3 loop sequence, consensus(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile-Gly-Y-(X)-Z

1.a. Complete V3 loop sequence, consensus(A)-(B)-(X)-Y-Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile-Gly-Asp-Ile-Arg-Gln-Ala-His-Cys-Y-(X)-Z

2. Partial V3 loop sequence, isolate HIV-1 SF2(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Argt-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Ile-Gly-Y-(X)-Z

3. Partial V3 loop sequence, isolate HIV-1 SC(A)-(B)-(X)-Y-Asn-Asn-Thr-Thr-Arg-Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Ala-Thr-Gly-Asp-Ile-Ile-Gly-Y-(X)-Z

4. Partial V3 loop sequence, isolate HIV-1 MN(A)-(B)-(X)-Y-Tyr-Asn-Lys-Arg-Lys-Arg-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly-Y-(X)-Z

5. Partial V3 loop sequence, isolate HIV-1 RF(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Thr-Lys-Gly-Pro-Gly-Arg-Val-Ile-Tyr-Ala-Thr-Gly-Gln-Ile-Ile-Gly-Y-(X)-Z

6. Partial V3 loop sequence, isolate HIV-1 mal(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Arg-Gly-Ile-His-Phe-Gly-Pro-Gly-Gln-Ala-Leu-Tyr-Thr-Thr-Gly-Ile-Val-Gly-Y-(X)-Z

7. Partial V3 loop sequence, isolate HTLV-IIIB(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Y-(X)-Z

8. Partial V3 loop sequence, isolate HIV-1 ELI(A)-(B)-(X)-Y-Gln-Asn-Thr-Arg-Gln-Arg-Thr-Pro-Ile-Gly-Leu-Gly-Gln-Ser-Leu-Tyr-Thr-Thr-Arg-Ser-Arg-Ser-Y-(X)-Z

9. Partial V3 loop sequence, isolate ANT70(A)-(B)-(X)-Y-Gln-Ile-Asp-Ile-Gln-Glu-Met-Arg-Ile-Gly-Pro-Met-Ala-Trp-Tyr-Ser-Met-Gly-Ile-Gly-Gly-Y-(X)-Z

10. Partial V3 loop sequence, Brazilian isolate, Peptide V3-368(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Arg-Gly-Ile-His-Met-Gly-Trp-Gly-Arg-Thr-Phe-Tyr-Ala-Thr-Gly-Glu-Ile-Ile-Gly-Y-(X)-Z

11. Carboxy-terminus, HIV-1 gp120(A)-(B)-(X)-Y-Arg-Asp-Asn-Trp-Arg-Ser-Glu-Leu-Tyr-Lys-Tyr-Lys-Val-Val-Lys-Ile-Glu-Pro-Leu-Gly-Val-Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Y-(X)-Z

2. Human immunodeficiency Virus type 2 Envelope Peptide

a. gp41, isolate HIV-2 rod

(A)-(B)-(X)-Y-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y-(X)-Z

b.

(A)-(B)-(X)-Y-Lys-Tyr-Leu-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y-(X)-Z

c. gp120, isolate HIV-2 NIHZ

(A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Leu-Pro-Ile-Thr-Phe-Met-Ser-Gly-Phe-Lys-Phe-His-Ser-Gln-Pro-Val-Ile-Asn-Lys-Y-(X)-Z

d. Partial V3 loop sequence, Peptide V3-GB12

(A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Val-Pro-Ile-Thr-Leu-Met-Ser-Gly-Leu-Val-Phe-His-Ser-Gln-Pro-Ile-Asn-Lys-Y-(X)-Z

e. Partial V3 loop sequence, Peptide V3-239

(A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Leu-Pro-Val-Thr-Ile-Met-Ser-Gly-Leu-Val-Phe-His-Ser-Gln-Pro-Ile-Asp-Asp-Y-(X)-Z

3. Chimpanzee immunodeficiency Virus

a. gp41

(A)-(B)-(X)-Y-Leu-Trp-Gly-Cys-Ser-Gly-Lys-Ala-Val-Cys-Y-(X)-Z

4. Simian immunodeficiency Virus

a. Transmembrane protein, isolate SIVagm (TY01)

(A)-(B)-(X)-Y-Ser-Trp-Gly-Cys-Ala-Trp-Lys-Gln-Val-Cys-Y-(X)-Z

b. Transmembrane protein, isolate SIVmnd

(A)-(B)-(X)-Y-Gln-Trp-Gly-Cys-Ser-Trp-Ala-Gln-Val-Cys-Y-(X)-Z

5. HTLV-I and HTLV-II Virus

Peptide I-gp46-3

(A)-(B)-(X)-Y-Val-Leu-Tyr-Ser-Pro-Asn-Val-Ser-Val-Pro-Ser-Ser-Ser-Ser-Thr-Leu-Leu-Tyr-Pro-Ser-Leu-Ala-Y-(X)-ZPeptide I-gp46-5

(A)-(B)-(X)-Y-Tyr-Thr-Cys-Ile-Val-Cys-Ile-Asp-Arg-Ala-Ser-Leu-Ser-Thr-Trp-His-Val-Leu-Tyr-Ser-Pro-X-(X)-Z

Peptide I-gp46-4

(A)-(B)-(X)-Y-Asn-Ser-Leu-Ile-Leu-Pro-Pro-Phe-Ser-Leu-Ser-Pro-Val-Pro-Thr-Leu-Gly-Ser-Arg-Ser-Arg-Arg-Y-(X)-Z

Peptide I-gp46-6

(A)-(B)-(X)-Y-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Ile-Trp-Phe-Leu-Asn-Thr-Glu-Pro-Ser-Gln-Leu-Pro-Pro-Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Y-(X)-Z

Peptide I-p21-2

(A)-(B)-(X)-Y-Gln-Tyr-Ala-Ala-Gln-Asn-Arg-Arg-Gly-Leu-Asp-Leu-Leu-Phe-Trp-Glu-Gln-Gly-Gly-Leu-Cys-Lys-Ala-Leu-Gln-Glu-Gln-Cys-Arg-Phe-Pro-Y-(X)-Z

Peptide I-p19

(A)-(B)-(X)-Y-Pro-Pro-Pro-Pro-Ser-Ser-Pro-Thr-His-Asp-Pro-Pro-Asp-Ser-Asp-Pro-Gln-Ile-Pro-Pro-Pro-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-Y-(X)-Z

Peptide II-gp52-1

(A)-(B)-(X)-Y-Lys-Lys-Pro-Asn-Arg-Gln-Gly-Leu-Gly-Tyr-Tyr-Ser-Pro-Ser-Tyr-Asn-Asp-Pro-Y-(X)-Z

Peptide II-gp52-2

(A)-(B)-(X)-Y-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Leu-Trp-Phe-Ile-Thr-Ser-Glu-Pro-Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Y-(X)-Z

Peptide II-gp52-3:

(A)-(B)-(X)-Y-Tyr-Ser-Cys-Met-Val-Cys-Val-Asp-Arg-Ser-Ser-Leu-Ser-Ser-Trp-His-Val-Leu-Tyr-Thr-Pro-Asn-Ile-Ser-Ile-Pro-Gln-Gln-Thr-Ser-Ser-Arg-Thr-Ile-Leu-Phe-Pro-Ser-Y-(X)-Z

Peptide II-p19

(A)-(B)-(X)-Y-Pro-Thr-Thr-Thr-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Pro-Glu-Ala-His-Val-Pro-Pro-Pro-Tyr-Val-Glu-Pro-Thr-Thr-Thr-Gln-Cys-Phe-Y-(X)-Z

These above-mentioned biotinylated peptides were synthesized and foundto be specifically recognized by antisera from infected humans orprimers are considered particularly advantageous. All theseabove-mentioned peptides are new.

The process of the invention enables to increase the antigenicity ofthese HIV peptides, which can however be bound to a support, even whenthey are not biotinylated.

The HCV peptide sequences which follow have been found to bespecifically recognized by antisera from infected humans or primates andwhich are considered particularly advantageous. The non-biotinylatedamino acid sequences can be synthesized according to classical methods.

The peptides of interest are intended to mimic immunologically proteinsor domains of proteins encoded by HCV. Since sequence variability hasbeen observed for HCV, it may be desirable to vary one or more aminoacids so as to better mimic the epitopes of different strains. It shouldbe understood that the peptides described need not be identical to anyparticular HCV sequence as long as the subject compounds are capable ofproviding for immunological competition with at least one strain of HCV.The peptides may therefore be subject to insertions, deletions andconservative as well as non-conservative amino acid substitutions wheresuch changes might provide for certain advantages in their use. Thepeptides will preferably be as short as possible while still maintainingall of the sensitivity of the larger sequence. In certain cases, it maybe desirable to join two or more peptides together into a singlestructure. The formation of such a composite may involve covalent ornon-covalent linkages.

Of particular interest are biotinylated peptides of HCV into whichcysteine, thioglycollis acid, or other thiol-containing compounds havebeen incorporated into the peptide chain for the purpose of providingmercapto-groups which can be used for cyclization of the peptides.

The following peptides from the Core region of HCV were determined ascorresponding to immunologically important epitopes.

1. Peptide I or Core 1 (aa. 1-20) has the following amino acid sequence:

(I)

(A)-(B)-(X)-Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Y-(X)-Z

2. Peptide II or Case 2 (aa. 7-26) has the amino acid sequence:

(II)

(A)-(B)-(X)-Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Y-(X)-Z

Of particular interest is the oligopeptide IIA (aa. 8 to 18):

(IIA)

(A)-(B)-(X)-Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Y-(X)-Z.

3. Peptide III or Core 3 (aa 13-32) has the sequence:

(III)

(A)-(B)-(X)-Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Y-(X)-Z

4. Peptide IV or Core 7 (aa 37-56) has the sequences:

(IV)

(A)-(B)-(X)-Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Y-(X)-Z

Of particular interest is the oligopeptide IVa or Core 6 (aa. 31 to 50):

(IVa)

(A)-(B)-(X)-Y-Val-Gly-Gly-Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Y-(X)-Z

5. Peptide V or Core 9 (aa 49-68) has the sequence:

(V)

(A)-(B)-(X)-Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Y-(X)-Z

Of particular interest is the oligopeptide Va (aa. 55 to 74):

(Va)

(A)-(B)-(X)-Y-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Y-(X)-Z

6. Peptide VI or Core 11 (aa 61-80) has the following sequence:

(VI)

(A)-(B)-(X)-Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Y-(X)-Z

7. Peptide VII (aa 73-92) or core 13 has the sequence:

(VII)

(A)-(B)-(X)-Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Y-(X)-Z

8. Peptide Core 123 (aa. 1-32):

(A)-(B)-(X)-Y-Met-Ser-Thr-Ile-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Y-(X)-Z

9. Peptide Core 7910 (aa. 37-80):

(A)-(B)-(X)-Y-Gly-Gly-Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Y-(X)-Z

The following peptides from the NS4 region of HCV were found tocorrespond to immunologically important epitopes.

Peptide VIII or NS4-1 or HCV1 (aa 1688-1707) has the sequence:

(VIII)

(A)-(B)-(X)-Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Y-(X)-Z

Peptide IX or HCV2 (aa 1694-1713) has the sequence:

(IX)

(A)-(B)-(X)-Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Y-(X)-Z

Peptide HCV3

(A)-(B)-(X)-Y-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Y-(X)-Z

Peptide X of HCV4 (aa 1706-1725) has the sequence:

(X)

(A)-(B)-(X)-Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Y-(X)-Z

11. Peptide XI or NS4-5 or HCV5 (aa 1712-1731) has the sequence:

(XI)

(A)-(B)-(X)-Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Y-(X)-Z

12. Peptide XII or HCV6 (aa 1718-1737) has the sequence:

(XII)

(A)-(B)-(X)-Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Y-(X)-Z

13. Peptide XIII or NS4-7 or HCV7 (aa 1724-1743) has the sequence:

(XIII)

(A)-(B)-(X)-Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Y-(X)-Z

14. Peptide XIV or HCV8 (aa 1730-1749) has the sequence:

(XIV)

(A)-(B)-(X)-Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Y-(X)-Z

15. Peptide NS4-27 or HCV9 (aa. 1712-1743):

(A)-(B)-(X)-Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Glu-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Y-(X)-Z

16. Peptide NS4e:

(A)-(B)-(X)-Y-Gly-Glu-Gly-Ala-Val-Gln-Trp-Met-Asn-Arg-Leu-Ile-Ala-Phe-Ala-Ser-Arg-Gly-Asn-His-Y-(X)-Z

The following peptides of the NS5 region of HCV were found to correspondto immunologically important epitopes.

Peptide XV or NS5-25 (aa 2263-2282) has the sequence:

(XV)

(A)-(B)-(X)-Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Y-(X)-Z

Peptide XVI or NS5-27 (aa 2275-2294) has the sequence:

(XVI)

(A)-(B)-(X)-Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Y-(X)-Z

Peptide XVII or NS5-29 (aa 2287-2306) has the sequence:

(XVII)

(A)-(B)-(X)-Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Y-(X)-Z

Peptide XVIII or NS5-31 (aa 2299-2318) has the sequence:

(XVIII)

(A)-(B)-(X)-Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Y-(X)-Z

Peptide XIX or NS5-33 (aa 2311-2330) has the sequence:

(XIX)

(A)-(B)-(X)-Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Y-(X)-Z

Peptide NS5-2527 (aa. 2263 to 2294):

(A)-(B)-(X)-Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asp-Tyr-Asn-Y-(X)-Z

The following peptides from the N-terminal region of the E2/NS1 regionof HCV were found to correspond to immunologically important epitopes.

peptide XXa (aa. 383-416)

(A)-(B)-(X)-Y-Gly-Glu-Thr-Tyr-Thr-Ser-Gly-Gly-Ala-Ala-Ser-His-Thr-Thr-Ser-Thr-Leu-Ala-Ser-Le-Phe-Ser-Pro-Gly-Ala-Ser-Gln-Arg-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXa-1 (aa. 383-404)

(A)-(B)-(X)-Y-Gly-Glu-Thr-Tyr-Thr-Ser-Gly-Gly-Ala-Ala-Ser-His-Thr-Thr-Ser-Thr-Leu-Ala-Ser-Leu-Phe-Ser-Y-(X)-Z

peptide XXa-2 (aa. 393-416)

(A)-(B)-(X)-Y-Ser-His-Thr-Thr-Ser-Thr-Leu-Ala-Ser-Leu-Phe-Ser-Pro-Gly-Ala-Ser-Gln-Arg-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXb (aa. 383-416)

(A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val-Ser-Gly-Gly-Ala-Ala-Ala-Ser-Asp-Thr-Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Pro-Gly-Ser-Ala-Gln-Lys-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXb-1 (aa. 383-404)

(A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val-Ser-Gly-Gly-Ala-Ala-Ala-Ser-Asp-Thr-Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Y-(X)-Z

peptide XXb-2 (aa. 393-416)

(A)-(B)-(X)-Y-Ala-Ser-Asp-Thr-Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Pro-Gly-Ser-Ala-Gln-Lys-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXc (aa. 383-416)

(A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val-Thr-Gly-Gly-Val-Gln-Gly-His-Val-Thr-Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Pro-Gly-Ala-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXc-1 (aa. 383-404)

(A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val-Thr-Gly-Gly-Val-Gln-Gly-His-Val-Thr-Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Y-(X)-Z

peptide XXc-2 (aa. 393-416)

(A)-(B)-(X)-Y-Gly-His-Val-Thr-Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Pro-Gly-Ala-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXc (aa. 383-416)

(A)-(B)-(X)-Y-Gly-His-Thr-His-Val-Thr-Gly-Gly-Arg-Val-Ala-Ser-Ser-Thr-Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Gln-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXd-1 (aa. 383-404)

(A)-(B)-(X)-Y-Gly-His-Thr-His-Val-Thr-Gly-Gly-Arg-Val-Ala-Ser-Ser-Thr-Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Y-(X)-Z

peptide XXd-2 (aa. 393-416)

(A)-(B)-(X)-Y-Ala-Ser-Ser-Thr-Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Gln-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn-Thr-Y-(X)-Z

peptide XXe (aa. 383-416)

(A)-(B)-(X)-Y-Gly-Asp-Thr-His-Val-Thr-Gly-Gly-Ala-Gln-Ala-Lys-Thr-Thr-Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Ser-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XXe-1 (aa. 383-404)

(A)-(B)-(X)-Y-Gly-Asp-Thr-His-Val-Thr-Gly-Gly-Ala-Gln-Ala-Lys-Thr-Thr-Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Y-(X)-Z

peptide XXe-2 (aa. 393-416)

(A)-(B)-(X)-Y-Ala-Lys-Thr-Thr-Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Ser-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XXf (aa. 383-416)

(A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr-Ser-Gly-Gly-Asn-Ala-Gly-His-Thr-Met-Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Pro-Gly-Pro-Lys-Gln-Asn-Val-His-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XXf-1 (aa. 383-404)

(A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr-Ser-Gly-Gly-Asn-Ala-Gly-His-Thr-Met-Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Y-(X)-Z

peptide XXf-2 (aa. 393-416)

(A)-(B)-(X)-Y-Gly-His-Thr-Met-Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Pro-Gly-Pro-Lys-Gln-Asn-Val-His-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XXg (aa. 383-416)

(A)-(B)-(X)-Y-Ala-Glu-Thr-Ile-Val-Ser-Gly-Gly-Gln-Ala-Ala-Arg-Ala-Met-Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Pro-Gly-Ala-Lys-Gln-Asn-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XXg-1 (aa. 383-404)

(A)-(B)-(X)-Y-Ala-Glu-Thr-Ile-Val-Ser-Gly-Gly-Gln-Ala-Ala-Arg-Ala-Met-Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Y-(X)-Z

peptide XXg-2 (aa. 393-416)

(A)-(B)-(X)-Y-Ala-Arg-Ala-Met-Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Pro-Gly-Ala-Lys-Gln-Asn-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XXh (aa. 383-416)

(A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr-Thr-Gly-Gly-Ser-Thr-Ala-Arg-Thr-Thr-Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Arg-Gly-Ala-Lys-Gln-Asp-Ile-Gln-Leu-Ile-Asp-Thr-Y-(X)-Z

peptide XXh-1 (aa. 383-404)

(A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr-Thr-Gly-Gly-Ser-Thr-Ala-Arg-Thr-Thr-Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Y-(X)-Z

peptide XXh-2 (aa. 393-416)

(A)-(B)-(X)-Y-Ala-Arg-Thr-Thr-Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Arg-Gly-Ala-Lys-Gln-Asp-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

The above-mentioned sequences correspond to epitopes localized on theHCV type-1 isolate HCV-1 (Choo et al. Proc; Natl. Acad. Sci. 88,2451-2455, 1991) and HC-J1 (Okamoto et al., Jap. J. Exp. Med. 60,167-177, 1990) sequence. It is, however, to be understood that alsopeptides from other type-1 HCV isolate sequences which correspond to theabove-mentioned immunologically important regions may also be comprisedin the composition according to the invention. An example of variant HCVsequences also falling within the present invention may be derived fromthe HCV-J isolate (Kato et al., Proc. Nat. Acad. Sci. 87, 9524-9528).

The following peptides derived from the same regions as the above-citedpeptide regions from the type 2 HCV sequences.

peptide XX/2

(A)-(B)-(X)-Y-Ala-Gln-Thr-His-Thr-Val-Gly-Gly-Ser-Thr-Ala-His-Asn-Ala-Arg-Thr-Leu-Thr-Gly-Met-Phe-Ser-Leu-Gly-Ala-Arg-Gln-Lys-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide XX/2-1

(A)-(B)-(X)-Y-Ala-Gln-Thr-His-Thr-Val-Gly-Gly-Ser-Thr-Ala-His-Asn-Ala-Arg-Thr-Leu-Thr-Gly-Met-Phe-Ser-Y-(X)-Z

peptide XX/2-2

(A)-(B)-(X)-Y-Ala-His-Asn-Ala-Arg-Thr-Leu-Thr-Gly-Met-Phe-Ser-Leu-Gly-Ala-Arg-Gln-Lys-Ile-Gln-Leu-Ile-Asn-Thr-Y-(X)-Z

peptide VIII-2 or NS4-1 (2)

(A)-(B)-(X)-Y-Val-Asn-Gln-Arg-Ala-Val-Val-Ala-Pro-Asp-Lys-Glu-Val-Leu-Tyr-Glu-Ala-Phe-Asp-Glu-Y-(X)-Z

peptide IX-2

(A)-(B)-(X)-Y-Val-Ala-Pro-Asp-Lys-Glu-Val-Leu-Tyr-Glu-Ala-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ala-Ser-Y-(X)-Z

peptide X-2

(A)-(B)-(X)-Y-Asp-Glu-Met-Glu-Glu-Cys-Ala-Ser-Arg-Ala-Ala-Leu-Ile-Glu-Glu-Gly-Gln-Arg-Ile-Ala-Y-(X)-Z

peptide XI-2 or NS4-5 (2)

(A)-(B)-(X)-Y-Ala-Ser-Arg-Ala-Ala-Leu-Ile-Glu-Glu-Gly-Gln-Arg-Ile-Ala-Glu-Met-Leu-Lys-Ser-Lys-Y-(X)-Z

peptide XII-2

(A)-(B)-(X)-Y-Ile-Glu-Glu-Gly-Gln-Arg-Ile-Ala-Glu-Met-Leu-Lys-Ser-Lys-Ile-Gln-Gly-Leu-Leu-Gln-Y-(X)-Z

peptide XIII-2 or NS4-7(2)

(A)-(B)-(X)-Y-Ile-Ala-Glu-Met-Leu-Lys-Ser-Lys-Ile-Gln-Gly-Leu-Leu-Gln-Gln-Ala-Ser-Lys-Gln-Ala-Y-(X)-Z

peptide XIV-2

(A)-(B)-(X)-Y-Ser-Lys-Ile-Gln-Gly-Leu-Leu-Gln-Gln-Ala-Ser-Lys-Gln-Ala-Gln-Asp-Ile-Gln-Pro-Ala-Y-(X)-Z

peptide XV-2

(A)-(B)-(X)-Y-Arg-Ser-Asp-Leu-Glu-Pro-Ser-Ile-Pro-Ser-Glu-Tyr-Met-Leu-Pro-Lys-Lys-Arg-Phe-Pro-(X)-Y-Z

peptide XVI-2

(A)-(B)-(X)-Y-Met-Leu-Pro-Lys-Lys-Arg-Phe-Pro-Pro-Ala-Leu-Pro-Ala-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Y-(X)-Z

peptide XVII-2

(A)-(B)-(X)-Y-Ala-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Ser-Trp-Lys-Arg-Pro-Asp-Tyr-Y-(X)-Z

peptide XVIII-2

(A)-(B)-(X)-Y-Glu-Ser-Trp-Lys-Arg-Pro-Asp-Tyr-Gln-Pro-Ala-Thr-Val-Ala-Gly-Cys-Ala-Leu-Pro-Pro-Y-(X)-Z

peptide XIX-2

(A)-(B)-(X)-Y-Val-Ala-Gly-Cys-Ala-Leu-Pro-Pro-Pro-Lys-Lys-Thr-Pro-Thr-Pro-Pro-Pro-Arg-Art-Arg-Y-(X)-Z

The above-mentioned sequences correspond to epitopes localized on theHCV type-2 isolate HC-J6 sequence (Okamoto et al., J. Gen. Virology 72,2697-2704, 1991). It is, however, to be understood that also peptidesfrom other type-2 HCV isolate sequences which correspond to theabove-mentioned immunologically important regions may also be comprisedin the composition according to the invention. Examples of variantsequences also falling within the present invention may be derived fromHCV isolate HC-J8 (Okamato et al., Virology 188, 331-341, 1992).

The following peptides from the NS4 region of HCV type 3 are alsopreferred peptides according to the present invention:

Peptide NS4-1 (3)

(A)-(B)-(X)-Y-Leu-Gly-Gly-Lys-Pro-Ala-Ile-Val-Pro-Asp-Lys-Glu-Val-leu-Tyr-Gln-Gln-Tyr-Asp-Glu-Y-(X)-Z

Peptide NS4-5 (3)

(A)-(B)-(X)-Y-Ser-Gln-Ala-Ala-Pro-Tyr-Ile-Glu-Gln-Ala-Gln-Val-Ile-Ala-His-Gln-Phe-Lys-Glu-Lys-Y-(X)-Z

Peptide NS4-7 (3)

(A)-(B)-(X)-Y-Ile-Ala-His-Gln-His-Gln-Phe-Lys-Glu-Lys-Val-Leu-Gly-Leu-Leu-Gln-Arg-Ala-Thr-Gln-Gln-Gln-Y-(X)-Z

It is to be understood that also other peptides corresponding to HCVtype-3 isolate sequences which correspond to immunologically importantregions as determined for HCV type-1 and type-2 may also be comprised inthe composition according to the invention.

The composition according to the present invention may also comprisehybrid HCV peptide sequences consisting of combinations of the coreepitopes of the HCV core (table 9) HCV NS4 (table 10) or the HCV NS5(table 11) region separated by Gly and/or Ser residues, andpreferentially the following hybrid HCV sequences:

Epi-152

(A)-(B)-(X)-Y-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Gly-Gly-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Gly-Gly-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Y-(X)-Z

Epi-33B3A

(A)-(B)-(X)-Y-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Gly-Gly-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Gly-Ser-Gly-Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Y-(X)-Z

Epi-4B2A6

(A)-(B)-(X)-Y-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Gly-Gly-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Ser-Gly-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Y-(X)-Z

The composition according to the present invention may also comprise socalled biotinylated mixotope sequences consisting of peptides containingat each position all the amino acids found in the naturally occurringisolates, with said peptides being derived from any of theabove-mentioned immunologically important regions (see FIG. 14).

(2) A preferred mixture of biotinylated peptides for detecting and/orimmunizing against Hepatitis C Virus, Human Immunodeficiency Virus type1 and Human Immunodeficiency Virus type 2 consists of:

A. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII, 1a.3, 1a.4, 1a.b,1b.1a, 2b, 2d,

B. II, III, IVa, Va, IX, IX-2, XI, XI-2, XIII, XIII-2, XV, XV-2, XVI,XVI-2, XVIII, XVIII-2, 1a.3, 1a.4, 1a.b, 1b.1a, 2b, 2d.

(3) A preferred mixture of biotinylated peptides for detecting and/orimmunizing against Human Immunodeficiency Virus types 1 and 2 and HumanLymphotropic Virus types I and II consists of:

1a.3, 1a.4, 1b.1, 2b, 2c, 2d, I-gp46-3, I-gp46-4, I-gp46-5, I-gp46-6,II-gp52-2, II-gp52-3, I-p21-2, I-p19, II-p19.

(4) Another preferred mixture of biotinylated peptides for detectingand/or immunizing against Hepatitis C Virus, Human ImmunodeficiencyVirus types 1 and 2 and Human Lymphotropic Virus types I and II consistsof:

1a.3, 1a.4, 1a.6, 1b.1a, 2d, II, III, IVa, Va, IX, XI, XIII, XV, XVI,XVIII, XXa-2, XXc-2, XXg-2, XXh-2, I-gp46-3, I-gp46-4, I-gp46-5,I-gp-46-6, II-gp52-3, I-p21-2, I-p19, II-p19.

(5) The present invention relates also to compositions of biotinylatedpeptides which are considered particularly advantageous, for diagnosticas well as immunogenic purposes for Hepatitis C Virus, and whichadvantageously comprise the following mixtures:

A. I, III, IVa, Va,

B. II, III, IVa, Va,

C. IX, XI, XIII,

D. XV, XVI, XVIII, XIX,

E. XXc-2, XXa-1, XXa-2, XXh-1, XXh-2, XXg-2, XX/2-2,

F. IX-2, XI-2, XIII-2,

G. XV-2, XVI-2, XVIII-2, XIX-2,

H. IX, IX-2, IX, XI-2, XIII, XIII-2,

I. XV, XV-2, XVI, XVI-2, XVIII, XVIII-2, XIX, XIX-2,

J. II, III, IVa, Va, IX, IX-2, XI, XI-2, XIII, XIII-2, XV, XV-2, XVI,XVI-2, XVIII, XVIII-2,

K. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII,

L. II, III, IV, V, IX, XI, XIII, XV, XVI, XVIII,

M. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII, XXa-2, XXc-2, XXg-2,XXh-2.

(6) The present invention relates also to compositions of biotinylatedpeptides which are considered particularly advantageous, for diagnosticas well as immunogenic purposes for Human Immunodeficiency Virus, andwhich are advantageously selected from the following mixtures: for type1:

A. 1a.3, 1a.4, 1a.5, 1a.b

B. 1a.3, 1a.4, 1b.1, 1b.3, 1b.6, 1b.10,

C. 1b.1, 1b.2, 1b.3, 1b.4, 1b.5, 1b.6, 1b.7, 1b.8, 1b.9, 1b.10

D. 1b.1, 1b.2, 1b.3, 1b.4, 1b.6, 1b.10,

E. 1a.3, 1a.4, 1a.5, 1a.b, 1b.1a, for type 2:

A. 2b, 2c, 2d, 2e, for types 1 and 2:

A. 1a.3, 1a.4, 1b.1, 2b, 2c, 2d,

B. 1a.3, 1a.4, 1b.1a, 2b, 2d.

(7) The present invention relates also to compositions comprisingbiotinylated peptides which are considered particularly advantageous,for diagnostic as well as immunogenic purposes for Human T-cellLymphotropic Virus and are advantageously selected from the followingmixtures:

for Human T-Lymphotropic virus type I:

Peptides I-gp46-1, I-gp46-4, I-gp46-5, I-gp46-6, I-p21-2, I-p19

for Human T-Lymphotropic virus type II:

Peptides II-gp52-1, II-gp52-2, II-gp52-3, I-gp46-4, II-p19, I-p21-2.

for Human lymphotropic virus types I and II:

Peptides I-gp46-3, I-gp46-4, I-gp46-5, I-gp46-6, II-gp52-1, IIgp52-2,II-gp52-3, I-p21-2, I-p19, II-p19.

The synthesis of the peptides may be achieved in solution or on a solidsupport. Synthesis protocols generally employ t-butyloxycarbonyl- or9-fluorenylmethoxycarbonyl-protected activated amino acids. Theprocedures for carrying out the synthesis, the amino acid activationtechniques, the types of side-chain production, and the cleavageprocedures used are amply described in, for example, Stewart and Young,Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company,1984; and Atherton and Sheppard, Solid Phase Peptide Synthesis, IRLPress, 1989.

(8) The present invention also relates to a process for in vitrodetermination of antibodies using the above defined biotinylatedpeptides, wherein said biotinylated peptides are preferably in the formof streptavidin-biotinylated peptide complexes or avidin-biotinylatedpeptide complexes.

In the complex of streptavidin-biotinylated peptides oravidin-biotinylated peptides, the peptides may be biotinylated eitherN-terminally, C-terminally or internally.

This approach for the determination of antibodies is not limited withrespect to peptide length and avoids the difficulties inherent incoating peptides directly onto the solid phase for immunologicalevaluation.

The use of biotinylated peptides, in the process of the invention, makesthe anchorage of peptides to a solid support such that it leaves theiressential amino acids free to be recognized by antibodies.

The expression anchoring peptide to a solid support means the attachmentof the peptide to a support via covalent bonds or non-covalentinteractions such that the peptide becomes immobilized.

The solid support can be nitrocellulose, polystyrene, nylon or any othernatural or synthetic polymer.

The expression “their essential amino acids are left free to berecognized by antibodies” means that amino acid side chains of thepeptide proper are neither chemically modified in any way nor involvedin the interaction between the peptide and the solid phase.

The use of biotinylated peptides in the process of the invention enablessaid biotinylated peptides to be free to assume a wide range ofconformations, among which at least one is appropriate for the bindingof antibodies to said biotinylated peptides.

Any biotinylated peptide can be selected to be used in the process ofthe invention. However, some of them are able to be anchored on solidsupport and to react with antibodies specifically recognizing theepitope within this peptide even without being biotinylated and withoutbeing involved in a complex of avidin of streptavidin. In this case, theuse of biotinylated peptides results in an apparent increase of theantigenicity of peptides with respect to the antigenicity observed whenthe peptides are not biotinylated. The expression “apparent” is meant toindicate an observed change obtained under similar test conditionswithout regard to the absolute cause of the observed change.

By “antigenicity” is meant the property of a peptide to be bound by anantibody.

By “increase of antigenicity” is meant that a positive signal isobtained for a dilution which is at least two times the dilution of thenon-biotinylated peptides. Said positive signal is of the same magnitudeas the one obtained for non-biotinylated peptides.

In other words, obtaining a positive signal can be obtained for asmaller amount of biotinylated peptide, compared to the amount ofnon-biotinylated peptide.

The present invention also illustrated a process for the identificationof epitopes in a protein sequence comprises the following steps:

the preparation of peptides corresponding to portions of the amino acidsequence of the protein or polypeptide to be analyzed, said peptidesbeing either contiguous, or preferably overlapping each other, theamount of overlapping being at least 3 amino acids, and preferably about6 to about 12, the length of the peptides being at least about 5 aminoacids and no more than about 50, preferably no more than about 40 aminoacids, and more preferably from 9 to about 30 amino acids, with saidpeptides being characterized in that they are biotinylated;

binding the peptides to a solid phase through the interaction betweenthe biotinyl group and streptavidin or avidin and measuring antibodybinding to the individual peptides using classical methods.

(9) The present invention also relates to a process for the in vitrodetermination of antibodies to HIV or diagnosis of HIV infection byusing a peptide composition as defined above in an immunoassayprocedure, wherein the biotinylated peptides used are in the form ofcomplexes of streptavidin-biotinylated or of avidin-biotinylatedpeptides.

(10) The present invention relates also to a process for the in vitrodetermination of antibodies to HCV or diagnosis of HCV infection byusing a peptide composition as defined above in an immunoassayprocedure, wherein the biotinylated peptides used are in the form ofcomplexes of streptavidin-biotinylated or of avidin-biotinylatedpeptides.

(11) The present invention relates also to a process for the in vitrodetermination of antibodies to HTLV I or II or diagnosis of HTLV I or IIinfection by using a peptide composition as defined above in animmunoassay procedure, wherein the biotinylated peptides used are in theform of complexes of streptavidin-biotinylated or of avidin-biotinylatedpeptides.

A preferred method for carrying out the in vitro determination ofantibodies is by means of an enzyme-linked immunosorbant assay (ELISA).This assay employs a solid phase which is generally a polystyrenemicrotiter plate or bead. The solid phase may, however, be any materialwhich is capable of binding a protein, either chemically via a covalentlinkage or by passive adsorption. In this regard, nylon-based membranesare also considered to be particularly advantageous. The solid phase iscoated with streptavidin or avidin and after a suitable period, excessunbound protein is removed by washing. Any unoccupied binding sites onthe solid phase are then blocked with an irrelevant protein such asbovine serum albumin or casein.

A solution containing the mixture or selection of biotinylated peptidesis subsequently brought into contact with the streptavidin- oravidin-coated surface and allowed to bind. Unbound peptide is removed bywashing. Alternatively, biotinylated peptides are allowed to formcomplexes with either avidin or streptavidin. The resulting complexesare used to coat the solid phase. After a suitable incubation period,unbound complex is removed by washing. An appropriate dilution of anantiserum or other body fluid is brought into contact with the solidphase to which the peptide is bound. The incubation is carried out for atime necessary to allow the binding reaction to occur. Subsequently,unbound components are removed by washing the solid phase. The detectionof immune complexes is achieved by using heterologous antibodies whichspecifically bind to the antibodies present in the test serum and whichhave been conjugated with an enzyme, preferably but not limited toeither horseradish peroxidase, alkaline phosphatase, or β-galactosidase,which is capable of converting a colorless or nearly colorless substrateor co-substrate into a highly colored product or a product capable offorming a colored complex with a chromogen which can be detectedvisually or measured spectrophotometrically.

Other detection systems known in the art may however be employed andinclude those in which the amount of product formed is measuredelectrochemically or luminometrically. The detection system may alsoemploy radioactively labeled antibodies, in which case the amount ofimmune complex is quantified by scintillation counting or counting. Inprinciple, any type of immunological test for the detection ofantibodies may be used, as long as the test makes use of the complexbetween either streptavidin or vidin and (a) biotinylated peptide(s)synthesized as described.

Also included are competition assays in which streptavidin- or avidin-biotinylated peptide complexes in solution are permitted to compete withthe solid phase-bound antigen for antibody binding or assays in whichfree peptide in solution is permitted to compete with solid phase-boundstreptavidin or avidin: biotinylated peptide complexes. By way ofexample, the many types of immunological assays for the detection andquantitation of antibodies and antigen are discussed in detail (Tijssen,P., Practice and Theory of Enzyme Immunoassays, Elsevier Press,Amsterdam, Oxford, N.Y., 1985).

The immunological assays may be restricted to single biotinylatedpeptides. Preferably, however, a mixture of biotinylated peptides isused which includes more than one epitope derived from the infectiousagent(s) to which the presence of specific antibodies is to be measured.

Another preferred method for carrying out the in vitro determination ofantibody detection is the line immunoassay (LIA).

This method of antibody detection consists essentially of the followingsteps:

the antigens, in the form of biotinylated peptide: streptavidin oravidin complexes, to be tested or used are applied as parallel linesonto a membrane which is capable of binding, covalently ornon-covalently, the antigen to be tested,

unoccupied binding sites on the membrane are blocked with an irrelevantprotein such as casein or bovine serum albumin,

the membrane is cut into strips in a direction perpendicular to thedirection in which the antigen (biotinylated peptide) lines are applied,

an appropriate dilution of an antiserum or other body fluid (containingantibodies to be detected) is brought into contact with a strip to whichthe antigens are bound and allowed to incubate for a period of timesufficient to permit the binding reaction to occur,

unbound components are removed by washing the strip,

the detection of immune complexes is achieved by incubating the stripwith heterologous antibodies which specifically bind to the antibodiesin the test serum and which have been conjugated to an enzyme such ashorseradish peroxidase,

the incubation is carried out for a period sufficient to allow bindingto occur,

the presence of bound conjugate is detected by addition of the requiredsubstrate or co-substrates which are converted to a colored product bythe action of the enzyme,

the reactions are detected visually or may be quantified bydensitometry.

(12) As demonstrated in the Examples section the present inventionrelates also the the use of a peptide composition as defined above, forimmunization against HIV, and/or HCV, and/or HTLV I or II infection.

(13) The present invention also relates to a method for preparing thebitinylated peptides used in the invention involves the use ofN-α-Fmoc-X (N-y-biotin) or N-α-Fmoc-X (N-y-biotin) derivative, wherein Xrepresents

where n is at least 1 but less than 10 and is preferably between 2 and6, one amino group being attached to the Cα atom while the other beingattached to carbon Cy, which is the most distal carbon in the sidechain; or their esters obtained with alcohol ROH and more particularlypentafluorophenyl ester;

y representing position y with respect to the carbon atom carrying theCOOH group in the radical.

This biotin derivative will be called intermediary product, and theabove-defined intermediary products are new compounds determinedaccording to the process of the invention.

(14) In an advantageous method for preparing the compounds of theinvention, the intermediary product can be represented by one of thefollowing formula:

N-α-Fmoc- (N-y-biotin) is N-α-Fmoc-lysine (ε-biotin) orN-α-Fmoc-ornithine (N-δ-biotin)

(15) The N-terminal biotinylated peptides can be prepared according tothe method which comprises the following steps:

addition of the successive amino acids duly protected onto the resin togive:

Fmoc−AA_(n) . . . AA₁−resin,

deprotection of the NH₂-terminal for instance by means of piperidine,

addition of the intermediary product:

 through its COOH onto the NH₂-terminal to obtain:

deprotection of the NH₂-terminal group of the compound obtained,cleavage from the resin, extraction and purification of the peptideobtained, biotinylated at its amino terminal, the steps of side chaindeprotection and peptide cleavage being liable to be performedsimultaneously or separately, and particularly

deprotection of the NH₂-terminal group of the intermediary group, forinstance by means of piperidine,

cleavage from the resin for instance with an acid such astrifluoroacetic acid, in the presence of scavengers such asethanedithiol, thioanisole, or anisole,

extraction of the peptide with a solvent such as diethylether to removemost the acid and scavengers,

purification, such as with HPLC to obtain:

Biotin can be conveniently coupled to the free amino-terminus of anotherwise fully protected peptide chain using also conventionalactivation procedures. Since biotin possesses one carboxyl group and noamino groups, biotin essentially functions as a chain terminator.Preferred activating agents for in situ activation include but are notlimited to benzotriazol-1-yl-oxo-tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), O-benzotriazol-1-yl-N, N, N′,N′-tetramethyluronium hexafluorophosphate (HBTU), andO-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU). The activation procedures employing these and related compoundsare known to those versed in the art of solid phase peptide synthesisand the coupling of biotin does not entail a significant departure fromstandard coupling protocols.

Biotin in a pre-activated form may also be used. EitherN-hydroxysuccinimidobiotin or bitinamidocaproate N-hydroxysuccinimideester are conveniently employed and both are commercially available.This method of coupling has been described by Lobl, T. J., Deibel, M.R., and Yem, A. W., Anal. Biochem. (1988) 170(2):502-511. Followingaddition of the N-terminal biotin, the peptide is cleaved from the resinin the presence of scavengers the choice of which will depend on theusual considerations of peptide amino acid composition and the nature ofthe protecting groups used.

(16) The carboxy terminal biotinylated peptides involved in the processof the invention can be prepared according to a method which comprises

coupling of a carboxy-activated form of the intermediary product asdefined above to a cleavable linker attached to the resin, for instanceto obtain the following compound:

deprotection of the α amino group of the intermediary compound, forinstance by means of piperidine to obtain:

successive addition of the subsequent amino acids AA₁ . . . AA_(n) dulyprotected onto

deprotection of the NH₂-terminal for instance by means of piperidine,

deprotection of the compound obtained, cleavage from the resin,extraction and purification of the peptide obtained, biotinylated at itscarboxy terminal end, the steps of side chain deprotection and peptidecleavage being liable to be performed simultaneously or separately, andparticularly

deprotection of the NH₂-terminal, for instance by means of piperidine,

cleavage from the resin for instance with trifluoroacetic acid, in thepresence of scavengers such as ethanedithiol, or thioanisole, oranisole,

extraction of the peptide with a solvent such as diethylether to removemost of the acid and scavengers,

purification, such as with HPLC to obtain:

(17) The internally biotinylated peptides can be prepared according to amethod which comprises the following steps:

addition of successive amino acids duly protected onto the resin togive:

Fmoc−AA_(n) . . . AA₁−resin,

deprotection of the NH₂-terminal,

addition of the intermediary product:

 through its COOH onto the NH₂-terminal to obtain:

deprotection of the α amino group of the intermediary compound, forinstance by means of piperidine to obtain:

addition of the subsequent amino acids duly protected onto the resin togive:

deprotection of the NH2 terminal group of the compound obtained,cleavage from the resin, extraction and purification of the peptideobtained, biotinylated at its amino-terminal, the steps of side chaindeprotection and peptide cleavage being liable to be performedsimultaneously or separately, and particularly,

deprotection of the NH₂-terminus, for instance by means of piperidine,

cleavage from the resin for instance with trifluoroacetic acid, in thepresence of scavengers such as ethanedithiol, or thioanisole, oranisole,

extraction of the peptide with a solvent such as diethylether to removemost of the acid and scavengers,

purification, such as with HPLC to obtain:

Under certain circumstances, it may prove particularly advantageous tobe able to biotinylate a peptide internally or at its carboxy-terminus.Such instances arise, for example, when the amino acid sequence of apeptide corresponds to the amino-terminal sequence of a protein.Attachment of a biotin to the amino-terminus of such a peptide resultsin a structure which is significantly different from that found in thenative protein and may, as a consequence, adversely affect the bindingproperties of biochemical properties of the peptide. It is also possiblethat even for peptides corresponding to internal protein sequences,their recognition by binding proteins or immunoglobulins may depend onwhich end of the peptide and the manner in which it is presented forbinding. The importance of peptide orientation has been described byDryberg, T. and Oldstone, M. B. A., J. Exp. Med. (1986) 164:1344-1349.

In order to be able to incorporate a biotinyl moiety into a peptide in aposition and sequence independent manner, efforts were made tosynthesize a suitable reagent which can be coupled using conventionalprocedures. A convenient reagent for C-terminal or internalbiotinylation is N-ε-biotinyl-lysine. Provided the α-amino group of thiscompound is suitably protected (Fmoc and tBoc), this reagent may be usedto introduce a biotin anywhere in the peptide chain, including at theamino terminus, by the standard procedures used in solid phase peptidesynthesis. The synthesis of the t-Boc-protected derivative has beendescribed (Bodansky, M., and Fagan, D T., J. Am. Chem. Soc. (1977)99:235-239) and was used to synthesize short peptides for use instudying the enzyme activities of certain transcarboxylases.

Unlike the t-Boc derivative, the synthesis of N-α-Fmoc-Lys (N-ε-biotin)has not been described and given the growing interest in Fmoc-basedsynthesis strategies, this compound is considered particularlyadvantageous.

There are a number of possible routes which can be taken to arrive atthe desired Fmoc-protected compound. These are shown in FIG. 1. In thefirst approach, commercially available N-α-Fmoc-Lys (N-ε-tBoc) can beused as the starting material. The N-ε-tBoc protection is removed usingtrifluoroacetic acid and a scavenger such as water. A slight molarexcess of the N-α-Fmoc-lysine so obtained is then reacted withcarboxy-activated biotin. The resulting product can be readily purifiedby selective extractions and standard chromatographic techniques. In analternative approach, N-α-Fmoc-Lys (N-ε-biotin) can be produced fromcommercially available N-ε-biotinyl lysine (biocytin) by reaction withfluorenylmethylsuccinimidyl carbonate. Numerous examples of thesereactions which can be used as guidelines are given in Atherton andSheppard, Solid Phase Peptide Synthesis, IRL Press, 1989.

The strategy shown in FIG. 1 (method A) may also be applied tosynthesize N-α-Fmoc-ornithine (N-δ-biotin) from commercially availableN-α-Fmoc-ornithine (N-δ-tBoc). The ornithine derivative differs from thelysine derivative only in the length of the side chain which, for theornithine derivative, is shorter by one carbon atom. The N-α-Fmoc-Lyscan be conveniently incorporated into the peptide chain using the samereagents for in situ activation described for free biotin.

Alternatively, N-α-Fmoc-Lys (N-ε-biotin)-O-pentafluorophenyl ester canbe conveniently synthesized from N-α-Fmoc-Lys (N-ε-biotin) andpentafluorophenyl trifluoroacetate using the base-catalyzedtransesterification reaction described by Green, M. and Berman, J.,Tetrahedron Lett. (1990) 31:5851-5852, for the preparation ofO-pentafluorophenyl esters of amino acids. This active ester can be useddirectly to incorporate N-α-Fmoc-Lys (N-ε-biotin) into the peptidechain. The class of above-defined intermediary products can be preparedaccording to a method which comprises the following steps:

reaction of a diamino-monocarboxylic acid previously described withfluorenylmethysuccinimidylcarbonate or fluorenylmethyl chloroformateunder conditions of carefully controlled pH to give the singly protectedN-α-Fmoc derivative,

or alternatively, use of commercially available N-α-Fmoc-protecteddiamino-monocarboxylic acids when the side chain amino group is providedwith a protecting group which is different from the Fmoc group used toprotect the α-amino group, the side chain amino group protection beingliable to be selectively removed under conditions which leave theN-α-Fmoc group intact,

purification of the mono-protected N-α-Fmoc-diamino-monocarboxylic acidderivative by selective extractions and chromatography,

reaction of the derivative obtained with a carboxy-activated derivativeof biotin, such as N-hydroxysuccinimide biotin, to obtain the(N-α-Fmoc)-(N-y-biotin) derivative which is the desired intermediaryproduct,

purification of the intermediary product by selective extractions,precipitations, or chromatography.

When the biotinylated peptides used in the process of the invention areto be provided with linker arms, these chemical entities may beconveniently attached to either the N- or C-terminus of a peptidesequence during solid phase synthesis using standard coupling protocols,as long as the amino groups of these compounds are provided withappropriate temporary amino group protection.

All these specific biotinylated peptides are new.

DESCRIPTION OF THE FIGURES

All the samples and sera mentioned in the figures and tables arerandomly chosen samples and sera, containing antibodies produced as aresult of naturally occurring infection by a viral agent.

FIG. 1 represents the strategies for the synthesis of N-α-Fmoc-lysine(N-ε-Biotin).

More particularly:

Method A corresponds to the synthesis of (N-α-Fmoc-Lys(N-ε-biotin) fromN-ε-Fmoc-Lys(N-ε-tBoc) and Method B corresponds to the synthesis of(N-α-Fmoc-Lys(N-ε-biotin) from N-ε-biotinyl lysine.

FIG. 2 represents the diagram obtained in reverse phase chromatographyof the precursors involved in the preparation of the intermediaryproducts defined above, and of the intermediary compounds.

The reverse phase chromatography has been carried out in the followingconditions:

gradient specifications:

buffer A: 0.1% TFA in H2O,

buffer B: 0.1% TFA in acetonitrile,

column: C2/C18 reverse phase (Pharmacia, Pep-S),

detection wavelength: 255 nanometers;

gradient:

0% B from 0 to 1 minute,

0% B to 100% B from 1 minute to 60 minutes,

0% B from 60 minutes to 70 minutes.

The first diagram corresponds to method A (see FIG. 1) and the seconddiagram corresponds to method B (see FIG. 1).

FIG. 3a represents the antibody binding to HCV peptide II (in an ELISA).

The upper left curve corresponds to sample 8320.

The upper right curve corresponds to sample 8242.

The lower left curve corresponds to sample 8243.

The lower right curve corresponds to sample 8318.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

The curve with crosses corresponds to non-biotinylated HCV peptide IIand the curve with dots corresponds to biotinylated HCV peptide II.

FIG. 3b represents the antibody binding to HCV peptide XI (in an ELISA).

The upper left curve corresponds to sample 8320.

The upper right curve corresponds to sample 8326.

The lower left curve corresponds to sample 8242.

The lower right curve corresponds to sample 8243.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

The curve with crosses corresponds to non-biotinylated HCV peptide XIand the curve with dots corresponds to biotinylated HCV peptide XI.

FIG. 3C represents the antibody binding to HCV peptide XVI (in anELISA).

The upper left curve corresponds to sample 8326.

The upper right curve corresponds to sample 8242.

The lower right curve corresponds to sample 8243.

The lower right curve corresponds to sample 8318.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

The curve with crosses corresponds to non-biotinylated HCV peptide XVIand the curve with dots corresponds to biotinylated HCV peptide XVI.

FIG. 4 corresponds to the detection of biotinylated peptides coateddirectly (in an ELISA).

The first curve corresponds to biotinylated HCV peptide II, the secondcurve to biotinylated HCV peptide XI and the third curve to biotinylatedHCV peptide XVI.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

FIG. 5 represents the structures of N- and C-terminally biotinylatedHIV-1 peptides (hereabove designated by 1a.1) originating from thetransmembrane (TM) protein of HIV-1.

FIG. 6a represents the detection of core epitopes in the Core region ofHCV using overlapping 9-mers (in an ELISA).

The sera used are indicated above each diagram.

The ordinates correspond to the optical density at 450 nm.

The abscissae correspond to the sequence of the protein in which thelocation of the epitope(s) is to be determined. For purposes of graphicillustration, the optical density is assigned to the first amino acid inthe respective nine-mer sequences.

FIG. 6b represents the detection of core epitopes in the NS4 region ofHCV using overlapping 9-mers (in an ELISA).

The sera used are indicated above each diagram.

The ordinates correspond to the optical density at 450 nm.

The abscissae correspond to the sequence of the protein in which thelocation of the epitope(s) is to be determined. For purposes of graphicillustration, the optical density is assigned to the first amino acid inthe respective nine-mer sequences.

FIG. 6c represents the detection of core epitopes in the NS5 region ofHCV using overlapping 9-mers (in an ELISA).

The sera used are indicated above each diagram.

The ordinates correspond to the optical density at 450 nm.

The abscissae correspond to the sequence of the protein in which thelocation of the epitope(s) is to be determined. For purposes of graphicillustration, the optical density is assigned to the first amino acid inthe respective nine-mer sequences.

FIG. 7a corresponds to the positions of biotinylated 20-mers withrespect to overlapping 9-mers (in an ELISA).

The abscissae corresponds to the protein sequence in which theepitope(s) is to be determined.

FIG. 7b corresponds to the positions of biotinylated 20-mers withrespect to overlapping 9-mers (in an ELISA).

The abscissae corresponds to the protein sequence in which theepitope(s) is to be determined.

FIG. 7c corresponds to the positions of biotinylated 20-mers withrespect to overlapping 9-mers (in an ELISA).

The abscissae corresponds to the protein sequence in which theepitope(s) is to be determined.

FIG. 8 represents a comparison of antibody recognition of biotinylatedand unbiotinylated HCV peptides by line immunoassay (LIA).

FIG. 9 represents a comparison of antibody recognition of biotinylatedcore peptides by line immunoassay (LIA).

The shorter and longer peptides are compared.

FIG. 10 represents an evaluation of type-specific HCV NS4 peptides byLine immunoassay (LIA).

FIG. 11 represents the amino acid sequence of peptides NS4-a to NS4-e.

FIG. 12 represents the composition of hybrid HCV peptides.

FIG. 13 represents the antibody recognition of hybrid HCV peptides.

FIG. 14 represents the construction scheme for mixotope peptides fromthe N-terminus of E2/NS1 of HCV type 1.

FIG. 15 represents the mixotope synthesis strategy.

FIG. 16 represents the synthesis of multiple antigen peptides (MAPs).

FIG. 17 represents the recognition of E2/NS1 peptides by sera fromrabbits immunized with E2/NS1 “b” peptide MAPs.

FIG. 18 represents the recognition of a commercially available serumpanel with a number of biotinylated HTLV-I and HTLV-II peptidesincorporated into LIA strips.

Table 1 represents the antibody recognition of unbiotinylated HIV-1 andHIV-2 peptides (designated by TM-HIV1 and TM-HIV-2) and biotinylatedHIV-1 and HIV-2 peptides (hereabove referred to as 1a.1 and 2a, and alsodesignated by TM-HIV-1 Bio and TM-HIV-2 Bio) in an ELISA.

Table 2 represents the comparison of antibody recognition ofunbiotinylated and biotinylated peptides from the V3 sequence of isolateHIV-1 mn (also referred as 1b.4) in an ELISA.

Table 3 represents the comparison of antibody recognition of thebiotinylated V3-mn peptide (referred to as 1b.4) bound to streptavidinand avidin, in an ELISA.

Table 4 represents the comparison of antibody recognition ofbiotinylated and unbiotinylated HCV peptides, in an ELISA.

More particularly:

Table 4A corresponds to the antibody binding to HCV peptide XI.

Table 4B corresponds to the antibody binding to HCV peptide XVI.

Table 4C corresponds to the antibody binding to HCV peptide II.

Table 4D corresponds to the antibody binding to HCV peptide III.

Table 4E corresponds to the antibody binding to HCV peptide V.

Table 4F corresponds to the antibody binding to HCV peptide IX.

Table 4G corresponds to the antibody binding to HCV peptide XVIII.

Table 5 represents a comparison of antibody binding to biotinylated andnon-biotinylated peptides, at different peptide coating concentrations,in an ELISA.

Table 6 represents the comparison of N- and C-terminally biotinylatedTM-HIV-1 peptide (referred to as 1a.1), in an ELISA.

Table 7 represents a comparison of antibody recognition ofunbiotinylated and carboxy-biotinylated HCV peptide I.

Table 8 represents the use of mixtures of biotinylated HIV and HCVpeptides for antibody detection, in an ELISA.

Table 9 represents sequences of the core epitopes of the HCV Coreprotein.

Table 10 represents sequences of the core epitopes of the HCV NS4protein.

Table 11 represents sequences of the core epitopes of the HCV NS5protein.

Table 12 represents the antibody binding of various Core, NS4, and NS5biotinylated 20-mers by 10 test sera.

Table 13 represents the antibody recognition of individual E2/NS1peptides (percent of all sera giving a positive reaction).

Table 14 represents the overall recognition of HIV V3-loop peptides.

Table 15 represents the recognition of HIV peptides according to thegeographical region.

Table 16 represents the recognition of European, African and BrazilianHIV-1-positive sera to HIV-I V3-loop peptides V3-con and V3-368.

Table 17 represents the recognition of HIV-2 positive sera to two HIV-2V3 loop peptides.

Table 18 represents the antibody recognition of hybrid peptides.

Table 19 represents the antibody recognition of mixed HTLV I and IIpeptides.

All amino acid sequences are given in the conventional and universallyaccepted three-letter code and where indicated in the one-letter code.The peptide sequences are given left to right which, by convention, isthe direction from the amino terminus to the carboxy-terminus.

A number of unconventional codes are also used to represent chemicalgroups or modifications and are defined as follows:

Group Code AC acetyl Bio D-biotinyl Fmoc 9-fluorenylmethoxycarbonyl tBoctertiary butyloxycarbonyl

EXAMPLE 1 Peptide Synthesis

All of the peptides described were synthesized on TentaGel S-RAM (RappPolymere, Tübingen, Germany), a polystyrene-polyoxyethylene graftcopolymerfunctionalized with the acid-labilelinker4-(α-Fmoc-amino-2′,4′-dimethoxybenzyl) phenoxyaceticacid (Rink,Tetrahedron Lett. (1987) 28:3787) in order to generate peptidecarboxy-terminal amides upon cleavage. t-Butyl-based side chainprotection and Fmoc-α-amino-protection was used. The guanidine-group ofarginine was protected with the 2,2,5,7,8-pentamethylchroman-6-sulfonylmoiety. The imidazole group of histidine was protected with either t-Bocor trityl and the sulfhydryl group of cysteine was protected with atrityl group. Couplings were carried out using preformedO-pentafluorophenyl esters except in the case of arginine where TBTU wasused as the activating agent in the presence of 1.5 equivalents of thebase N-methylmorpholine. Occasionally, glutamine and asparagine werealso coupled using TBTU activation. In these cases, the trityl-protectedderivatives of these amino acids were employed. Biotin was coupled usingeither TBTU or HBTU. All syntheses wee carried out on a Milligen 9050PepSynthesizer (Novato, Calif.) using continuous flow procedures.Following cleavage with trifluoroacetic acid in the presence ofscavengers and extraction with diethylether, all peptides were analyzedby C18-reverse phase chromatography.

EXAMPLE 2 Synthesis of N-α-Fmoc-Lys (N-ε-Biotin)

A. Method A

Commercially available N-α-Fmoc-L-lysine (N-ε-tBoc) (1.5 grams) wastreated with 20 milliliters of 95% trifluoroacetic acid, 5% H₂O for 2hours at room temperature. Most of the acid was then evaporated under astream of nitrogen. Ten milliliters of water was added and the solutionwas extracted 3 times with diethylether. The aqueous phase was thenevaporated to dryness in vacuo over phosphorus pentoxide. The resultingpowder (N-α-Fmoc-L-lysine) was analyzed by reverse phase chromatographyand revealed a homogeneous product which was, as expected, morehydrophilic than the starting material.

N-α-Fmoc-lysine (190 mg, 0.49 mmol) was dissolved in 8 milliliters of0.1 M borate buffer, pH 8.7. N-hydroxysuccinimidobiotin (162 mg, 0.47mmol) was dissolved in 4 milliliters of dimethylformamide and added tothe solution of N-α-Fmoc-lysine. The pH was monitored and titrated asnecessary, with NaOH. After 2 hours, the solution was acidified with HClto pH 2.0, at which time a white precipitate was obtained.

Following extraction with ethylacetate and centrifugation, the whiteprecipitate was found at the H2O: ethylacetate interface. Both phaseswere removed and the precipitate extracted twice with 10 mM HCl, oncewith ethylacetate, followed by two extractions with diethylether. Theprecipitate was dissolved in DMF and precipitated by addition ofdiethylether. The crystalline powder was then dried in vacuo overphosphorus pentoxide. The resulting product was analyzed by reversephase chromatography and revealed a major peak which, as expected,eluted later than N-α-Fmoc-Lys. A very small peak of N-α-Fmoc-Lys wasalso observed. (FIG. 2a).

B. Method B

Commercially available N-ε-biotinyl lysine (biocytin, Sigma, 249 mg,0.67 mmol) was dissolved in 8 milliliters of 1 M Na2CO3 and cooled onice. Fluorenylmethylsuccinimidyl carbonate (222 mg, 0.66 mmol) wasdissolved in 2 milliliters of acetone and was added to the biotinyllysine solution over a period of 30 minutes with vigorous stirring.Stirring was continued for 5 hours at room temperature. The pH wasmaintained between 8 and 9 by addition of 1 M Na₂CO₃ as necessary. Theacetone was then evaporated off under vacuum, and 1.0 M HCl was addeduntil the pH of the solution was approximately 2. Upon acidification ofthe solution, a white precipitate appeared which was washed twice with10 mM HCl, twice with ethyl acetate, and twice with diethylether. Theprecipitate was dissolved in DMF and precipitated by addition ofdiethylether. The crystalline powder was then thoroughly dried in vacuoover phosphorus pentoxide. The resulting product was analyzed by reversephase chromatography and revealed a major peak which eluted with thesame retention time (30.5 minutes) as the product obtained using method1 (FIG. 2b).

EXAMPLE 3 Methods for the Determination of Peptides Corresponding toImmunologically Important Epitopes in an Enzyme-linked ImmunosorbentAssay (ELISA) Using Specific Antibodies

Where peptides were to be coated directly, stock solutions of thepeptides were diluted in sodium carbonate buffer, pH 9.6 and used tocoat polystyrene microtiter plates at a peptide concentration of 2 to 5micrograms per milliliter for 1 hour at 37° C.

In cases where biotinylated peptides were to be evaluated, plates werefirst coated with streptavidin in sodium carbonate buffer, pH 9.6 at aconcentration of 3 micrograms per milliliter for 1 hour at 37° C. Theplates were then washed to remove excess, unbound protein. A workingsolution of the biotinylated peptide at 1 microgram per milliliter insodium carbonate buffer was then added to the wells of the microtiterplate and incubated for 1 hours at 37° C.

Once the plates had been coated with antigen, any remaining free bindingsites on the plastic were blocked with casein. After washing, a dilutionof the appropriate antisera, usually 1:100, was added to the wells ofthe plates and incubated for 1 hour at 37° C.

After washing to remove unbound material, specific antibody binding wasdetected by incubating the plates with goat anti-human immunoglobulinantibodies conjugated to the enzyme horseradish peroxidase. Followingremoval of unbound conjugate by washing, a solution containing H₂O₂ and3,3′,5,5′-tetramethylbenzidine was added.

Reactions were stopped after a suitable interval by addition of sulfuricacid. Positive reactions gave rise to a yellow color which wasquantified using a conventional microtiter plate reader. Absorbancemeasurements were made at a wavelength of 450 nanometers and all dataare expressed as an optical density value at this wavelength.

EXAMPLE 4 Use of Biotinylated HIV Peptides for the Detection ofHIV-specific Antibodies

Experiments were performed to evaluate antibody recognition of short, 10amino acid-long, N-acetylated peptides corresponding to other containedwithin the transmembrane proteins of HIV-1 and HIV-2. Direct coating ofthese peptides in the wells of microtiter plates gave very poor resultswhen antibody binding was evaluated in an ELISA. Since it was suspectedthat the peptides did not bind well to the polystyrene solid phase, thepeptides were resynthesized in the same way except that biotin wasattached to the amino terminus of the peptides, separated from thedecamer peptide sequence by three glycine residues whose function it wasto serve as a linker arm. The peptides used for the comparison were asfollows:

TM-HIV-1:

Ac-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-NH2

TM-HIV-1 Bio

Bio-Gly-Gly-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-NH2

TM-HIV-2

Ac-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-NH2

TM-HIV-2 Bio

Bio-Gly-Gly-Gly-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-NH2

The biotinylated peptides were loaded onto microtiter plates which hadbeen coated with streptavidin. Antibody binding to these peptides wascompared to antibody binding to the unbiotinylated peptides which werecoated directly onto microtiter plates. The results are shown inTable 1. It is evident that the biotinylated peptides from the HIV-1 orHIV-2 transmembrane proteins bound to streptavidin are recognized verywell by antisera from HIV-1 or HIV-2 infected persons respectively. Thisis in contrast to the unbiotinylated versions of these peptides coateddirectly onto the polystyrene plates. Addition control experimentsshowed that the increase in antibody binding was the result of thespecific interaction between the biotinylated peptide and streptavidin,since there was no difference in antibody recognition of thebiotinylated or unbiotinylated peptides when both were coated directlyonto the microtiter plate.

Some peptides, particularly ones which are 15 amino acids in length orlonger, bind sufficiently to the solid phase to allow the detection ofspecific antibodies which recognize (an) epitope(s) present in thepeptide sequence.

To ascertain whether biotinylated would also improve antibodyrecognition of longer peptides, both the biotinylated and unbiotinylatedversions of the partial V3 loop sequence of isolate HIV-1 mn weresynthesized. The sequence and method of synthesis of both peptides wereidentical except at the amino terminus. The unbiotinylated peptide wassimply acetylated whereas in the biotinylated version, two glycineresidues were added as a linker arm to separate the peptide from thebiotinyl moiety.

The sequences of the two peptides used are as follows:

unbiotinylated V3 mn peptide

Ac-Tyr-Asn-Lys-Arg-Lys-Arg-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly-NH2,

biotinylated V3 mn peptide (peptide 1b.4)Bio-Gly-Gly-Tyr-Asn-Lys-Arg-Lys-Arg-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly-NH2.

The unbiotinylated peptide was coated directly onto the wells of apolystyrene microtiter plate while the biotinylated peptide was bound towells which had previously been coated with streptavidin. The resultsshown in Table 2 demonstrate that antibody binding to the biotinylatedpeptide is superior to antibody binding to peptide coated directly ontothe plastic.

EXAMPLE 5 Use of Biotinylated Peptides—Avidin Complexes for AntibodyDetection

Having demonstrated that antibody recognition of this peptide isimproved when the peptide is biotinylated and bound to streptavidin, anadditional experiment was performed to determine whether streptavidincould be substituted by avidin. The results shown in Table 3 indicatethat this is the case and that biotinylated peptides bound to avidin arerecognized very efficiently by specific antibodies.

EXAMPLE 6 Use of Biotinylated HCV Peptides for Detection of HCV SpecificAntibodies

In order to determine whether the enhanced antibody recognition ofbiotinylated peptides was a general phenomenon, a number of additionaltwenty amino acid-long peptides were synthesized which correspond tosequences derived from the hepatitis C virus (HCV) polyprotein. Theamino acid sequences evaluated were as follows:

a. HCV peptide XI

Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys

b. HCV peptide XVI

Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn

c. HCV peptide II

Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly

d. HCV peptide III

Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly

e. HCV peptide V

Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val

f. HCV peptide IX

Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln

g. HCV peptide XVIII

Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro

In each case, two versions of the peptide were synthesized. In theunbiotinylated version, the peptide was acetylated at the aminoterminus. The biotinylated versions were all N-terminally biotinylated.A linker arm consisting of two glycine residues separated the biotinylmoiety from the amino acids comprising the HCV sequence.

The unbiotinylated peptides were adsorbed onto the wells of polystyrenemicrotiter plates at a concentration of 3 micrograms per milliliter.

The biotinylated peptides were bound at a concentration of 1 microgramper milliliter to streptavidin-coated microtiter plates. Sera known tocontain antibodies to these peptides were used for the evaluation andwere tested to a 20-fold dilution. The results of these comparisons areshown in Table 4, a to g.

These results clearly indicate that antibody recognition of biotinylatedpeptides bound to streptavidin is enhanced relative to that of peptidescoated directly onto the wells of the microtiter plate.

EXAMPLE 7 Influence of Coating Concentration on Antibody Detection

To investigate further the enhanced antibody recognition of biotinylatedHCV peptides bound to streptavidin or avidin as compared to directadsorption on plastic, the influence of peptide coating concentrationwas investigated. Three peptides (HCV peptides II, XI, and XVI) werecoated in concentrations ranging from 10 nanograms per milliliter to 3micrograms per milliliter in a volume of 200 microliters per microtiterplate well. For direct coating, the unbiotinylated versions of thesepeptides were used. The biotinylated versions of these peptides wereused to coat wells to which streptavidin had previously been adsorbed.Sera known to contain antibodies to these peptides were used at adilution of 1 to 100 to evaluate the magnitude of antibody binding.

The numerical results of this experiment are shown in Table 5 and aredepicted graphically in FIG. 3, a-c.

It is evident that with few exceptions, the biotinylated peptide isrecognized very well even at the lowest concentration tested (10nanograms per milliliter, 2 nanograms per well). In many cases, opticaldensity values close to the maximum attainable are observed at a peptideconcentration of only 30 nanograms per milliliter (6 nanograms perwell). In contrast, however, the unbiotinylated peptides adsorbeddirectly onto the plastic are poorly bound by antibody, if at all.

EXAMPLE 8 Influence of Biotinylation of Peptides on Coating Efficiencyof the Peptides on a Solid Phase

To determine if the absence of a signal was due to lack of peptideadsorption when the peptides were coated directly, an additionalexperiment was performed. In this case, the biotinylated versions of thepeptides were coated directly onto the plastic at the sameconcentrations used in the previous experiment for the unbiotinylatedversions. To ascertain whether biotin-labeled peptide was bound, themicrotiter plates were incubated with a streptavidin: horseradishperoxidase conjugate. Since each peptide contains a single biotinylgroup, the resulting optical densities are a measure of the amount ofpeptide bound, although the absolute amount of bound peptide is notknown. The results presented graphically in FIG. 4 demonstrate thatplastic-bound peptide can be detected. As expected, the curves aredifferent for each peptide which is a reflection of their chemicaluniqueness. Two of the peptides, HCV peptides XI and XVI, appear to bindonly weakly to the wells of the polystyrene microtiter plate and thispoor binding is reflected in the low optical density values obtained inthe ELISA. Since the binding of the biotinylated peptides tostreptavidin-coated wells results in very good antibody recognition, itis obvious that poor binding of the peptide to the solid phase is not alimitation when use is made of interaction between biotin andstreptavidin.

On the other hand, one of the peptides, HCV peptide II, shows verysignificant binding to the solid phase, particularly at higher coatingconcentrations. However, at no coating concentration did the signalobtained when the peptide was coated directly ever equal the signalobtained when the biotinylated peptide was bound to streptavidin. Sinceeven at the lowest concentration tested, the streptavidin-boundbiotinylated versions of this peptide clearly gives a positive signalwith the antisera tested, the results would seem to indicate either thatthe direct coating of this peptide is extraordinarily inefficient orthat other factors are important besides the simple binding of peptideto the solid phase.

Although difficult to quantify, one of the factors almost certainlyinvolves the manner in which the peptide is bound and available forantibody binding. In the case of peptides coated directly onto the solidphase, it is virtually inevitable that some proportion of the peptidemolecules will interact with the solid phase through amino acid sidechains which are also essential for antibody recognition. These peptidemolecules will therefore be unable to participate in the bindingreaction with antibodies. This problem is not encountered with thebiotinylated peptides which are all bound to the solid phase through theinteraction between biotin and the solid phase-bound streptavidin.

EXAMPLE 9 Use of C-terminally Biotinylated HIV Peptides for SpecificAntibody Recognition

In order to determine whether the peptides biotinylated at theircarboxy-terminus also give use to enhanced antibody recognition, acarboxy-biotinylated version of the TM-HIV-1 peptide was synthesized.N-α-Fmoc-Lys (N-ε-biotin) prepared by method A as described was coupleddirectly to resin functionalized with the acid labile linker4-(α-Fmoc-amino-2′,4′-dimethoxybenzyl) phenoxyacetic acid after removalof the linker-bound Fmoc group with 20 percent piperidine. The couplingwas performed using a 3-fold molar excess of N-α-Fmoc-Lys (N-ε-biotin)relative to resin functional groups. Carboxyl group activation wasachieved using one equivalent of HBTU, one equivalent of1-hydroxybenzotriazole and 1.5 equivalents of N-methylmorpholine.N-methyl morpholine was dispensed as a 0.6 M solution indimethylformamide containing 40 percent dimethylsulfoxide which wasnecessary to achieve complete dissolution of the N-α-Fmoc-Lys(N-ε-biotin). Inspection of the Fmoc deprotection peak followingcoupling of the N-α-Fmoc-Lys (N-ε-biotin) indicated that coupling hadproceeded smoothly and efficiently. Two glycine residues were coupled toseparate the biotinyl lysine from the TM-HIV-1 amino acid sequence.Following synthesis of the peptide, the amino terminus was acetylatedwith acetic anhydride. The resulting structure of thecarboxy-biotinylated peptide differs significantly from the peptidebiotinylated at the amino terminus. A comparison of these structures isshown in FIG. 5.

In order to evaluate antibody recognition of these two peptides, thepeptides were bound individually to streptavidin-coated microtiterplates and tested using a panel of antisera from HIV-1 seropositivedonors. The results of this comparison is shown in Table 6. Clearly,antibody recognition of the C-terminally biotinylated peptide comparesvery favorably with that of the N-terminally biotinylated peptide. Theseresults also confirm the utility of the reagent N-α-Fmoc-Lys(N-ε-biotin) for carboxy-terminal biotinylation.

EXAMPLE 10 Comparison of Antibody Recognition of HCV Peptide I, CoatedDirectly (Unbiotinylated) or Bound to Streptavidin-coated Plated(Carboxy-terminal Biotinylation)

A similar experiment was performed using a peptide which bindsrelatively well to polystyrene ELISA plates in order to determinewhether the carboxy-biotinylated form of the peptide would result insuperior antibody recognition relative to the unbiotinylated form of thepeptide. The peptide chosen was HCV peptide I, which was synthesized inthe following versions:

a. unbiotinylated version:

H2N-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-CONH2

b. carboxy-biotinylated version:

H2N-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Gly-Gly-Lys(Bio)-CONH2.

A spacer consisting of two glycine residues was added at thecarboxy-terminus to physically separate the HCV portion of the peptideproper from the Lys(N-ε-Bio). Synthesis was performed on resinfunctionalized with 4-(α-Fmoc-amino-2′,4′-dimethoxybenzyl) phenoxyaceticacid linker in order to generate carboxy-terminal amides upon cleavage.Coupling of the N-α-Fmoc-Lys-(N-ε-biotin) to the linker was performedusing a 3-fold molar excess of the intermediate product relative to thelinker. Activation of the N-α-Fmoc-Lys(N-ε-biotin) was achieved usingone equivalent of TBTU, one equivalent of 1-hydroxybenzotriazole, and1.5 equivalents of N-methylmorpholine. The coupling of all other aminoacids was performed according to conventional protocols. Followingcleavage of the peptides in trifluoroacetic acid in the presence of theappropriate scavengers, the peptides were precipitated and extractedwith diethylether.

Unbiotinylated HCV peptide I was coated directly onto the wells of apolystyrene ELISA plate at a concentration of 3 micrograms permilliliter in sodium carbonate buffer, pH 9.6. Biotinylated HCV peptideI was bound to streptavidin-coated wells using a stock solutioncontaining the peptide at a concentration of 1 microgram per milliliter.The resulting plates were then incubated in parallel with a panel ofsera from HCV-seropositive donors. The results of this comparison areshown in Table 7. The biotinylated peptide clearly gives superiorresults relative to the unbiotinylated version of the same sequence. Twoof the sera (8326 and 8244) recognize the biotinylated version of thispeptide far better than the unbiotinylated version. The specificity ofthe antibody reaction is also reflected by the low optical densityvalues obtained for 5 serum samples from uninfected donors (F88, F89,F76, F136, and F6).

EXAMPLE 11 Use of Mixtures of Biotinylated HIV and HCV Peptides

In many cases, the use of mixtures of peptides is required to give thedesired result. Mixtures of peptides may be used for the detection ofantibodies directed against one or more proteins of a single virus, orfor the detection of antibodies directed against proteins of severalviruses in a single test. Such tests are considered particularlyadvantageous for the screening of blood donations for their suitabilityfor use in transfusions and as a source of blood products. In suchcases, ELISA plates or other solid supports coated with suitablemixtures of peptides may be used to screen samples for the presence ofantibodies to one or more infectious agents whose presence would renderthe sample unsuitable for use. For the diagnosis of specific infectiousagents, appropriate mixtures of peptides are required in order to obtainaccurate determinations. Antibodies to individual viral antigens derivedfrom one or more infectious agents may be individually detected andidentified simultaneously when use is made of test systems in whichindividual peptides or mixtures of peptides are bound to the solid phasebut are physically separated as they are, for example, in the lineimmunoassay, such that individual reactions can be observed andevaluated. Such tests require the use of an appropriate combination ofpeptide mixtures to achieve the desired result.

It is frequently preferable to use mixtures of peptides rather than asingle peptide for the diagnosis of ongoing or past infections. Sinceindividual responses to single epitopes may be quite variable, morereliable results are often obtained when several immunologicallyimportant epitopes are present in the antibody test. However, since eachpeptide is chemically unique, it is frequently difficult to incorporateall of the desired peptides into one test, particularly when thepeptides are to be coated directly onto the solid phase. Not allpeptides are capable of binding to the solid phase and the peptides inthe mixture may also exhibit very different optimal coating conditionsin terms of pH, ionic strength, and buffer composition.

To determine how well biotinylated peptides would function in a mixturewhen bound to streptavidin- or avidin-coated plates, two mixtures weremade of the N-terminally biotinylated versions of the HIV-1 peptidesTM-HIV-1 (hereabove referred to as 1a.1) and V3-mn (hereabove referredto as 1b.4), the HIV-2 peptide TM-HIV-2 (hereabove referred to as 2a),and the hepatitis C virus peptides II, IX, and XVIII. Mixture Acontained each of the six biotinylated peptides at a concentration of 1microgram per milliliter (6 micrograms per milliliter peptide, total)while in mixture B, each peptide was present at a concentration of 0.1microgram per milliliter (0.6 microgram per milliliter peptide, total).The individual peptides were coated at a concentration of 1 microgramper milliliter. For purposes of comparison, mixtures A and B were alsocoated directly onto the wells of a microtiter plate. Samples fromHIV-1, HIV-2, and HCV-seropositive donors were tested and compared tosera from seronegative blood donors. A cut-off absorbance value of 0.250was used to determine whether a reaction was positive or negative.Absorbance values equal or greater than 0.250 were considered positivewhile absorbance values below this value were considered negative. Theresults of this experiment are shown in Table 8.

Based on the reactions to the individual peptides, all of the HCV serumsamples were negative for antibodies to either HIV-1 or HIV-2. One HIV-2sample (no. 1400) had antibodies to HCV peptide XVIII. Of the HIVsamples tested, there was no indication of cross reactivity and theELISA based on individual peptides is specific.

Both mixtures A and B gave good results when bound to avidin-coatedmicrotiter plates. As expected, these mixtures were recognized by HIV-1,HIV-2, and HCV-positive sera but not by sera from seronegative blooddonors. In contrast, when these mixtures were coated directly onto themicrotiter plates, the results were considerably less satisfactory, withmany samples giving a reaction which fell below the cut-off valueapplied. These results serve to illustrate quite convincingly theenhanced immunological recognition of biotinylated peptides bound toavidin as compared to peptides coated directly onto the solid phase aswell as the advantages of using mixtures of peptides for multipleantibody detection.

EXAMPLE 12 Use of Biotinylated Peptides for Mapping of Epitopes inDiagnostically Useful Regions of HCV

It was demonstrated in Example 6 that several diagnostically importantregions of the HCV polyprotein, such as Core, NS4, and NS5, can beidentified using overlapping 20-mer biotinylated peptides. Extensiveserological testing identified the most useful 20-mer biotinylatedpeptides which permitted to develop a line immunoassay utilizing thesebiotinylated peptides. However, it was desirable to know more exactlywhere in these 20 amino acid-long sequences the epitopes were located.One reason is that, if shorter sequences could be identified, it wouldbe possible to make synthetic peptides containing two or three epitopeswithout the peptide becoming prohibitively long.

Epitopes present in a position of the putative HCV proteins were mappedusing the method originally described by Geysen, H. M., Meloen, R. H.,and Barteling, S. J.; Proc. Natl. Acad. Sci. USA (1984) 81:3998-4002.Consecutive peptides nine amino acids in length with an eight amino acidoverlap were synthesized on polyethylene pins derivatized with anon-cleavable linker. This peptide length was chosen because it islarger than the size of typical linear epitopes which are generallybetween 5 and 7 amino acids in length. By synthesizing 9-mers, theprobability that epitopes would be missed was minimized.

The regions in the HCV polyprotein which were scanned contain Coresequences (aa. 1 to 80), NS4 (aa. 1688 to 1755), and NS5 (aa. 2191 to2330). These regions correspond to the previously determined 20-mers:Peptide I to VII (Core 1 to 13), Peptide VIII to XIV (NS4-1 to 9), andpeptide XV to XIX (NS5-13to 33).

Following synthesis, all peptides were N-acylated prior to side chaindeprotection in order to remove the unnatural positive charge at theamino terminus.

The peptides were then assayed for their ability to be recognized byantibodies present in sera from HCV seropositive donors. The results ofthese experiments are shown in FIGS. 6a to 6 b. The optical densityvalues shown are the average of duplicate determinations and have beenassigned to the first amino acid of the 9-mer sequence.

The antibody binding profiles for 10 different HCV sera are shown inFIG. 6a. It is clear that the core protein of HCV presents well-definedlinear epitopes which are readily stimulated by synthetic peptides. Atleast superficially, most sera appear to give very similar patterns.Closer inspection, however, reveals that there are individualdifferences. The various regions of the HCV core protein which arerecognized by antibodies are perhaps more properly termed epitopicclusters rather than epitopes as such, since each region is undoubtedlycomposed of several overlapping epitopes which are difficult, if notimpossible, to distinguish using polyclonal sera. An attempt was made toidentify core epitopes in each of the epitopic clusters. Used in thissense, the word “core” refers to the minimal amino acid sequencerecognizable by antibodies. It should be emphasized, however, that aminoacids in addition to the core sequence may improve reactivityparticularly in the case of polyclonal sera. An analysis of the epitopesis given in Table 9. By comparing the reactions of the various sera,subdomains of epitopic clusters could be identified. Some sera reactpredominantly with one subdomain and not with others, while other serarecognize all of the subdomains but still allow the subdomains to bedistinguished because each forms a shoulder in the large peak whichdefines that particular epitopic clusters. Table 9 and FIG. 7a shows thelocations of the core epitopes with respect to the sequences of the20-mers.

The series of 9-mers corresponding to each of the 20-mer Core peptidesare shown in FIG. 7a together with the placement of each of thesesequences in relation to an antibody recognition profile for one of theantisera tested.

The antigenic profiles for the NS4 protein obtained with the 10 sera areshown in FIG. 6b. In general, the reaction of these sera with the 9-merswas less pronounced than with the peptides from the Core protein. Itwas, nevertheless, still possible to identify epitopic regions in theN-terminal sequences of the viral NS4 protein. The core sequences ofthese epitopes are analyzed in Table 10 and show their relation to the20-mer synthetic peptides which are diagnostically important in thisregion. The 9-mers corresponding to the different 20-mers are shown inFIG. 7b together with their placement in relation to an example of anantigenic profile. It can be seen that the 20-mers correspond quite wellto the epitopes in this region.

The portion of the NS5 protein which was scanned corresponds to theregion covered by the 20-mer peptides 13 to 33. The antigenic profilesobtained in this region are shown in FIG. 6c. Again, an attempt was madeto define core epitopes and these are listed in Table 11. Littleantibody binding was observed in the amino terminal portion of thissequence. In FIG. 7c, the 9-mers corresponding to the 20-mer peptidesNS5-21 to NS5-31 are listed and their positions are shown relative toone of the antigenic profiles.

In particular, it is apparent that, the importance of the sequencerepresented by HCV peptide XVI (NS5-27) would be severely underestimatedbased on the results obtained with the overlapping 9-mers. Theimportance of this sequence would also be underestimated ifunbiotinylated HCV peptide XVI (NS5-27) were evaluated in an ELISAfollowing direct coating onto the microtiter plate (see Table 4B).However, the biotinylated version of this peptide when bound tostreptavidin- or avidin-coated plates reveals the presence of a veryimportant epitope which is of diagnostic value.

In contrast to the often weak binding observed with the 9-mers, thebinding with the 20-mers was frequently quite strong (see table 12). Inseveral cases the differences are dramatic. For example, serum 8241 doesnot recognize any of the 9-mers, whereas the binding to the peptidesHCV2 (peptide IX) and HCV5 (peptide XI) is very strong. Moderate bindingwas also observed to the peptide HCV7 (peptide XIII). This would seem toindicate that there is an important structural component to theseepitopes which is present in the 20-mers but which is absent in the9-mers.

EXAMPLE 13 Use of Biotinylated Peptides for Identification of Epitopesin the N-terminus of NS1 Region of HCV Line Immunoassay

Epitopes can also be identified using the line immunoassay (LIA). Ingeneral, unbiotinylated peptides bind better to nylon membranes than topolystyrene ELISA plates. Nevertheless, biotinylated peptides complexedwith streptavidin or avidin give superior results in the lineimmunoassay than do their unbiotinylated counterparts bound directly tothe membrane. In order to illustrate this, unbiotinylated andN-terminally biotinylated versions of HCV peptides XXg-1 and XXg-2 weresynthesized. The unbiotinylated peptides were applied to the membrane asa stock solution containing 100 micrograms per milliliter peptide,whereas the biotinylated peptides were bound to streptavidin and appliedas a stock solution of 100 micrograms per milliliter complex. The amountof biotinylated peptide in the stock solution was thereforeapproximately 10 micrograms per milliliter. Three human IgG controllines were also applied to the strips in order to assist in evaluatingthe intensity of the reactions. Following application of the antigenlines, excess binding sites on the membrane were blocked with casein inphosphate-buffered saline. The membrane was subsequently cut into stripsperpendicular to the direction in which the antigen lines were appliedand the resulting strips were incubated with a panel of sera fromHCV-seropositive donors. Bound antibody was detected visually using goatanti-human IgG antibodies conjugated to the enzyme alkaline phosphataseafter addition of 5-bromo-4-chloro-3-indolylphosphate and Nitro Bluetetrazolium. The results are shown in FIG. 8.

The specificity of the reactions is demonstrated by the absence ofdetectable antibody binding to any of the HCV peptides by three sera(33, 34, and 35) obtained from HCV-seronegative donors. The reactions ofsera 1 to 32 to the unbiotinylated HCV peptides XX-1 and XX-2 aregenerally absent or exceedingly weak. In contrast, many of the seratested recognized the biotinylated versions of these peptides whencomplexed to streptavidin. The antibody reactions to the biotinylatedpeptides are significantly stronger in spite of the fact that onlyapproximately one-tenth the amount of peptide was present in these stocksolutions compared to the amount present in the stock solutions of theunbiotinylated peptides. The results obtained using the biotinylatedpeptides demonstrate the presence of a diagnostically useful epitope inthese peptide sequences which is not evident when the unbiotinylatedversions of the peptides are used.

A total of 8 sequences spanning the hypervariable N-terminus of the HCVE2-NS1 region (aa 383 to 416 of the HCV polyprotein) of different HCVisolates were chosen for further evaluation. These aligned sequences(One-letter code) are as following:

XXa GETYTSGGAASHTTSTLASLFSPGASQRIQLVNT (1)

XXb GHTRVSGGAAASDTRGLVSLFSPGSAQKIQLVNT (2)

XXc GHTRVTGGVQGHVTCTLTSLFRPGASQKIQLVNT (3)

XXd GHTHVTGGRVASSTQSLVSWLSQGPSQKIQLVNT (4)

XXe GDTHVTGGAQAKTTNRLVSMFASGPSQKIQLINT (5)

XXf AETYTSGGNAGHTMTGIVRFFAPGPKQNVHLINT (6)

XXg AETIVSGGQAARAMSGLVSLFTPGAKQNIQLINT (7)

XXh AETYTTGGSTARTTQGLVSLFSRGAKQDIQLINT (8)

These sequences are derived from isolates described by the followinggroups:

(1) Hijikata et al., Biochem. Biophys. Res. Comm. 175:220-228, 1991.

(2) unpublished results

(3) Hijikata et al., Biochem. Biophys. Res. Comm. 175:220-228, 1991.

(4) Kato et al., Proc. Natl. Acad. sci. USA 87:9524-9528, 1990.

(5) Takamizawa et al., J. Virology 65:1105-1113, 1991.

(6) Weiner et al., Virology 180:842-848, 1991.

(7) Okamoto et al., Japan. J. Exptl. Med. 60:167-177, 1990.

(8) Kremsdorfl et al., Abstract V64, Third International Symposium onHCV, Strasbourg, France, September, 1991.

Since the sequences are rather long and because secondarystructure—related difficulties were predicted to occur during synthesis,it was decided to split the sequences into two overlapping parts(“a”=amino acids 383 to 404 and “b”=amino acid 393 to 416 of the HCVpolyprotein).

XXaa GETYTSGGAASHTTSTLASLFS (SEQ ID NO: 406)

XXab SHTTSTLASLFSPGASQRIQLVNT (SEQ ID NO: 407)

XXba GHTRVSGGAAASDTRGLVSLFS (SEQ ID NO: 408)

XXbb ASDTRGLVSLFSPGSAQKIQLVNT (SEQ ID NO: 409)

XXca GHTRVTGGVQGHVTCTLTSLFR (SEQ ID NO: 410)

XXcb GHVTCTLTSLFRPGASQKIQLVNT (SEQ ID NO: 411)

XXda GHTHVTGGRVASSTQSLVSWLS (SEQ ID NO: 412)

XXdb ASSTQSLVSWLSQGPSQKIQLVNT (SEQ ID NO: 413)

XXea GDTHVTGGAQAKTTNRLVSMFA (SEQ ID NO: 414)

XXeb AKTTNRLVSMFASGPSQKIQLINT (SEQ ID NO: 415)

XXfa AETYTSGGNAGHTMTGIVRFFA (SEQ ID NO: 416)

XXfb GHTMTGIVRFFAPGPKQNVHLINT (SEQ ID NO: 417)

XXga AETIVSGGQAARAMSGLVSLFT (SEQ ID NO: 418)

XXgb ARAMSGLVSLFTPGAKQNIQLINT (SEQ ID NO: 419)

XXha AETYTTGGSTARTTQGLVSLFS (SEQ ID NO: 420)

XXhb ARTTQGLVSLFSRGAKQDIQLINT (SEQ ID NO: 421)

Subdividing the sequence also allows the position of the epitopes to bemore accurately defined.

All of the peptides were N-terminally biotinylated, complexed withstreptadivin and used to prepare LIA-strips (data not shown).

When only the LIA-positive samples are considered, the detection rate onthe E2/NS1 peptides was found to be on the order of 90 percent. Thecorrelations between recognition of the E2/NS1 peptides and LIAreactivity as well as the scores for the individual peptides are shownin Table 13. It was also clear from the observed reactions that theprimary epitope in this sequence is located towards the carboxyterminusof the hypervariable region. There wer exceptions to this, however. Eachserum appeared to have its own recognition pattern which underscores theimportance of using a mixture of different sequences if this epitope isto be included as a line in the LIA. It would also appear that eitherthere is a considerable degree of crossreactivity between the type 1aand type 1b sequences, or that most people are doubly infected. It is asimple matter to distinguish between these two possibilities byselectively removing the antibodies which bind to one sequence andlooking to see what the effect is on antibody recognition of the othersequences. A number of samples gave a rather weak reaction to one ormore E2/NS1 peptides but were LIA negative. While most probably falsepositive reactions, these sera may also be from people who wherepreviously infected but who have resolved the infection.

EXAMPLE 14 Use of Combined HCV Peptides from the Core Region of HCV forthe Detection of Antibodies by LIA

In order to reduce the overall number of peptides in a HCV ELISA or LIA,biotinylated peptides can be synthesize which span other immunologicallyimportant peptides. Examples of such “combined” HCV peptides from thecore protein NS3 region of HCV are given below:

Sequence Core 1 (I) MSTIPKPQRKTKRNTNRRPQ (SEQ ID NO: 133) Core 2 (II)    PKPQRKTKRNTNRRP (SEO ID NO: 134) Core 3 (III)            RNTNRRPQDVKFPGGGQIVG (SEQ ID NO: 135) Core 123MSTIPKPQRKTKRNTNRRPQDVKFPGGGQIVG (SEQ ID NO: 136) Core 6 (IVa)VGGVYLLPRROPRLGVRATR (SEO ID NO: 137) Core 7 (IV)      LPRRGPRLGVRATRKTSERS (SEQ ID NO: 138) Core 9 (V)                  TRKTSERSQPRGRRQPIPKV (SEQ ID NO: 139) Core 10 (VI)                        RSQPRGRRQPIPKVRRPEGR (SEO ID NO: 140) Core 7910 GGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKVRR (SEQ ID NO: 141)

All of these peptides have been provided with a Gly—Gly spacers and abiotin at the amino terminus. The peptides were evaluated in a lineimmunoassay experiment (LIA) and compared to the shorter core peptides.The results are shown in FIG. 9. The longer core peptides compare veryfavorably to the shorter peptides and consistently give a more intensereaction. This is could be explained if (i) the longer peptidesincorporate two or more epitopes which were previously spread over twoseparate peptides and/or (2) there is any conformational contributionwhich may be more prominent in the longer peptides.

EXAMPLE 15 Use of Combined HCV Peptides from the NS4 and NS5 Regions ofHCV for the Detection of Antibodies by LIA

Other peptides combine sequences in NS4 and NS5 which are as following(SEQ ID NOs:142-147, respectively):

Peptide Sequence NS4-5 (XI) S Q H L P Y I E Q G M M L A E Q F K Q KNS4-7 (XIII)                         L A E Q F K Q K A L G L L Q T A S RQ A NS4-57 S Q H L P Y I E Q G M M L A E Q F K Q K A L G L L Q T A S R QA NS5-25XV) E D E R E I S V P A E I L R K S R R F A NS5-27 (XVI)                        L R K S R R F A Q A L P V W A R P D Y N NS5-2527E D E R E I S V P A E I L R K S R R F A Q A L P V W A R P D Y N

The general advantage in using the longer peptides lies in the fact thattheir use in an ELISA or LIA leaves more space for the incorporation ofother peptides carrying immunologically important epitopes.

EXAMPLE 16 Use of Type-specific HCV NS4 Peptides for the Detection ofAntibodies by LIA

Equivalent peptides containing HCV type 2 and type 3 NS4 sequences whichcorrespond to the type 1 peptides found to contain epitopes in NS4 weresynthesized. The sequences of these peptides are shown below forcomparison (SEQ ID NOs:154-162, respectively):

Peptide Sequence NS4-1 (1) L S G K P A I I P D R E V L Y R E F D E NS4-1(2) V N Q R A V V A P D K E V L Y E A F D E NS4-5 (1) S Q H L P Y I E QG M M L A E Q F K Q K NS4-5 (2) A S R A A L I E E G Q R I A E M L K S KNS4-7 (1) L A E Q F K Q K A L G L L Q T A S R Q A NS4-7 (2) I A E M L KS K I Q G L L Q Q A S K Q A

LIA strips were prepared using these nine peptides which weresubsequently incubated with different sera. The results are shown inFIG. 10. Two of the sera which were previously negative on type 1 NS4peptides gave a positive reaction to the type 3 and type 2 peptides.This indicates that it is possible to increase the NS4 detection rateusing these peptides.

EXAMPLE 17 Use of Biotinylated Peptides from the V3 Loop Region of GP120of Different HIV-1 Isolates in a Line Immunoassay for the Detection ofHIV Antibodies

In order to determine the general diagnostic value of the V3 loop regionof gp120, nine peptides derived from this region of nine different HIV-1isolates were synthesized and included in a LIA. All nine peptides wereprovided with a Gly—Gly spacer and an N-terminal biotin. The alignedpeptides (one-letter amino acid code) sequences are as following:

CON NNTRKSIHI--GPGRAFYTTGEIIG 23 SF2 NNTRKSIYI--GPGRAFHTTGRIIG 23 SCNNTTRSIHI--GPGRAFYATGDIIG 23 MN YNKRKRIHI--GPGRAFYTTKNIIG 23 RFNNTRKSITK--GPGRVIYATGQIIG 23 MAL NNTRRGIHF--GPGQALYTTG-IVG 22 BHNNTRKSIRIQRGPGRAFVTIGKI-G 24 ELI QNTRQRTPI--GLGQSLYTT-RSRS 22 ANT70QIDIQEMRI--GP-MAWTSMG-IGG 21

The peptides were mixed with streptavidin in a slight molar excess overbiotin binding sites and the peptide:streptavidin complexes wereseparated from unbound material over Sephadex G-25. Material eluting inthe void volume was used in the preparation of the LIA.

A total of 332 sera were tested which had been obtained from variousgeographical regions. Since it is known that virus strains isolated inEurope or North America exhibit less strain-to-strain variability thanAfrican isolates, geographical differences in the V3-loop sequencerecognition were to be expected. The reactions of the various lines wereevaluated as positive (i) or negative (o) (data not shown).

A complete evaluation of the sera is given in Table 14. In total, 307 ofthe 332 sera gave a reaction to at least one peptide on the V3-loop LIA.Those sera which failed to give a reaction to any peptide on the V3-loopLIA were tested by Western Blot to determine whether the sera wereindeed positive for anti- HIV-I antibodies. It was found that 6 serawere in fact negative. The total number of positive sera tested wastherefore 326. There were, however, 19 sera which contained antibodiesto gp120 which failed to react with any of the V3-loop LIA, thepercentage of sera giving a positive reaction was, in global terms, 94%.There were, however, significant geographical differences. Thesedifferences are shown in Table 15.

The total percentage of sera from the different geographical regionsgiving at least one positive reaction can be summarized as follows:

European 100%  African 94% Brazilian 92%

Additional evaluations with European samples indicate that thispercentage is, in fact, some what less than 100% (data not shown).African samples which failed to give a reaction in the LIA have not beentested by Western Blot to confirm the presence of other HIV antibodies.

That the European sera would score well as expected. The lower scoreobtained for the African sera was also not totally unexpected, since itis known that there is more viral heterogeneity in Africa. Since V3-loopsequences of African strains of HIV have not been as extensivelycharacterized as the European or North American strains, it is clearthat we either do not have a representative sequence, or that attemptingto characterize African strains in terms of a consensus sequence is notpossible exercise since there is too much sequence divergence. Theresults obtained with the Brazilian sera were unexpected since nothinghas ever been reported concerning HIV variability in Brazil. From theseresults, it appears that the situation in Brazil more closely resemblesthe situation in Africa and not the situation in North America orEurope.

EXAMPLE 18 Improved Detection of HIV-1 Anti-V3 Domain Antibodies inBrazilian Sera Using a V3 Sequence Derived From a Brazilian Isolate.

Brazilian serum samples which failed to recognize any HIV-1 V3 loopsequences present on the previously described LIA strips but which werepositive for antibodies which recognized the HIV-1 gp120 protein onWestern blots were selected for further study. In one of these samples,V3 loop sequences of virus present in the serum sample could beamplified using the polymerase chain reaction using primers derived fromthe more constant regions flanking the hypervariable domain. Theresulting DNA fragment was subsequently cloned and the nucleotidesequence was determined. A peptide corresponding to the deduced aminoacid sequence encoded by this fragment was synthesized and tested forits ability to be recognized by various HIV-1 antibody-positive sera.The sequence of this peptide was as follows:

Peptide V3-368:

Asn Asn Thr Arg Arg Gly Ile His Met Gly Trp Gly Arg Thr Phe Tye Ala ThrGly Glu Ile Ile Gly

A spacer consisting of two glycine residues was added to the aminoterminus. Thereafter, the resulting N-terminal glycine residue wasbiotinylated. The ability of European, African, and Brazilian HIV-1antibody-positive sera to recognize this peptide was investigated andcompared to the ability of these same sera to recognize the consensussequence peptide in an ELISA. The two peptides were also evaluatedtogether as a mixture. These results are summarized in table 16. Theseresults demonstrate that with sera of European or African origin, theV3-368 peptide does not result in an increased anti-V3 loop antibodydetection over that which is observed with the V3con peptide. Incontrast, the use of the V3-368 peptide results in a marked improvementin V3 antibody detection with Brazilian sera. Although this peptide isrecognized less frequently than the V3con peptide, the two peptidescomplement each other to raise the detection rate from 83.1 percentusing the V3con peptide alone to 97.2 percent when the two peptides areused together.

EXAMPLE 19 Antibody Recognition of HIV-2 V3 loop sequences

The outer membrane glycoprotein of HIV-2 (gp105) is similar to that ofHIV-1 with respect to its organization. Like the gp120 protein of HIV-1,the gp105 protein of HIV-2 consists of domains of variable sequenceflanked by domains of relatively conserved amino acid sequence. In orderto detect antibodies specific for the V3 domain of HIV-2 produced inresponse to infection by this virus, biotinylated peptides weresynthesized corresponding to the V3 sequences of the HIV-2/SIV isolatesGB12 and isolate SIV mm 239 (Boeri, E., Giri, A., Lillo, F. et al.; J.Virol. (1992) 66(7):4546-4550). The sequences of the peptidessynthesized are as follows:

V3-GB12:

Asn Lys Thr Val Val Pro Ile Thr Leu Met Ser Gly Leu Val Phe His Ser GlnPro Ile Asn Lys

V3-239:

Asn Lys Thr Val Leu Pro Val Thr Ile Met Ser Gly Leu Val Phe His Ser GlnPro Ile Asn Asp

Two glycine residues were added at the N-terminus of each peptide toserve as a spacer and a biotin was coupled to the α-amino group of theresulting N-terminal glycine. The peptides were bound to streptavidinand coated in the wells of microwell plates. HIV-2 antibody-positivesera were used to evaluate these two peptides in an ELISA. These resultsare summarized in Table 17. The results clearly demonstrate theusefulness of these two peptide sequences for the diagnosis of HIV-2infection.

EXAMPLE 20 Localization of the Epitope at the Carboxy Terminus of C-100with Biotinylated Peptides

There have been various reports of an epitope located towards thecarboxy-terminal portion of the C-100 protein (EP-A-0 468 527, EP-A-484787). Reactivity of certain sera toward this epitope and not to epitopeslocated within the 5-1-1 fragment could explain why these sera give apositive reaction on C-100 but not to the above-described peptidesdescribed in the above-mentioned examples. The five overlappingbiotinylated peptides synthesized NS4-a, b, c, d and e are shown in FIG.11 and cover the carboxy-terminus of C-100 except for the last threeamino acids. LIA strips prepared with these peptides were tested using aseries of HCV Ab-positive and negative sera. The results of thisexperiment (data not shown) are summarized below:

Peptide Nr. of reactive sera Percentage NS4-a 0 0% NS4-b 2 3% NS4-c 0 0%NS4-d 0 0% NS4-e 16 27%

EXAMPLE 21 Use of Biotinylated Hybrid Peptides containing Epitopes fromdifferent HCV proteins

A fine mapping of the epitopes in the immunologically most importantregions of the HCV polyprotein using 9-mers was performed as illustratedin Example 12. Using this information, 3 peptide sequences were devisedwhich consisted of three 9-mer stretches of HCV sequence separated by 2amino acid-long spacers. In general, Gly-Gly, Gly-Ser or Ser-Gly spacerswere used to provide chain flexibility. The arrangement of the epitopesin the three hybrid peptides synthesized and their sequences are shownin FIG. 12. The three peptides were evaluated on a LIA strip. In thefirst evaluation, the sera originally used for the epitope fine mappingexperiments were used since the precise interactions of these sera withthe epitopes is known. These results are shown in FIG. 13 and aresummarized in Table 16. The order in which the epitopes wereincorporated into these three hybrid peptides was arbitrary. It isadvantageous, however, to link the epitopes together in a limited numberof peptide chains rather than attempting to develop a test based onindividual 9-mers. The use of separate 9-mers would rapidly saturate thestreptavidin binding sites on the plate (one biotin binding site/9-mer)whereas incorporating the 9-mers into a limited number of peptides aswas done in these experiments would enable one to bind 3 times as much(one biotin binding site/three 9-mers).

EXAMPLE 22 E2/NSl “b” Sequence Mixotope Peptides

The results using synthetic peptides (see Examples above) have indicatedthat most HCV seropositive sera contain antibodies directed towards thehypervariable N-terminus of E2/NSl. However, because of thehypervariable nature of this region of the protein, it is necessary touse a rather wide spectrum of sequences in order to detect theseantibodies in an acceptably high percentage of sera. Analysis ofavailable sequences revealed that the observed amino acid substitutionswere not entirely random and that certain amino acids were preferred incertain positions within the sequence. Since the hypervariable sequenceis rather long, this sequence who divided into two overlapping portions(“a” and “b”) to improve the quality of the product and simplify thesynthesis. Subdividing this region also permitted the determination ofthat the portion of this N-terminal segment of the E2/NSl protein whichwas most frequently recognized by antibodies was located in the regionencompassed by the “b” versions of these sequences. Given the sequenceinformation shown in FIG. 14 a “mixotope” was synthesized which containsat each position all the amino acids found in the naturally occurringisolates examined. The strategy followed in the synthesis of themixotope is depicted in FIG. 15. The strategy for designing mixotopes isreviewed in Gras-masse et al., Peptide Res. (1992) 5:211-216. The resinwas divided into a number of portions equal to the number of amino acidsto be coupled. The coupling reactions were carried out individually soas to avoid problems arising due to differences in coupling kineticsbetween the various amino acids. Following the coupling reactions, theresin portions were pooled and mixed thoroughly. The total number ofvariants obtained for this 23 amino acid-long sequence was +1.147×1010.The increasing number of variants as a function of chain length asmeasured from the carboxy-terminus or amino-terminus is shown in FIG.14. The rationale behind the mixotope approach is that epitopes arecomposed of amino acids whose contribution to antibody binding is notequal. Antibodies may recognize an epitope even though these may be arelatively large number of (generally not random) substitutions incertain positions. In this respect, the antigenic complexity of themixotope should be substantially less than the number of variantscomprising the mixture. For the sake of illustration, if it is assumedthat an average epitope is 6 amino acids in length, it is possible tocalculate the number variants for each successive 6 amino acid longsegment in the sequence. The number of variants as a function ofposition in the sequence is shown in FIG. 14. The actual number offunctional variant sequences will be equal to the number shown for any 6amino acid-long sequence which happens to correspond to an epitope,divided by a degeneracy factor equal to the number if toleratedsubstitutions in each position of the epitope but modified to reflectthe degree to which the particular substitutions are tolerated.Unfortunately, the exact position(s) of the epitope(s) are not known. Itshould be stated explicitly that this is not a random peptide library.Key positions in the total sequence which do not tolerate substitutions,as evidenced by the absence of amino acid variations in naturallyoccurring isolates, are preserved. One disadvantage of this syntheticapproach is that rare amino acid substitutions are overrepresented andwill tend to dilute out the more commonly encountered amino acids. Onthe other hand, the possibility existed that overrepresentation of raresubstitutions might allow the detection of antibodies not detectablewith epitope sequences comprised of more frequently encountered aminoacids. Following completion of the synthesis of the mixotope, allpeptide chains were provided with a (Gly)2 spacers and a biotin tofacilitate immunological evaluation. A multiple antigen peptide (MAP)version of the mixotope may also be synthesized in parallel.

One result of previous studies was that while approximately 90 percentof HCV-positive sera could e shown to contain anti-E2/NSl antibodiesdirected against the N-terminal hypervariable region with the 16 “a” and“b” sequences investigated. The apparent lack of these antibodies in theremaining 10 percent of HCV antibody-positive sera could be due to twofactors: 1) these patients fail to produce antibodies against thisportion of E2/NSl, or 2) has not yet been identified the correctsequence with which to detect these antibodies. Based on experimentswith the HIV-1 V3 loop, this latter possibility did not seem at allunrealistic. LIA strips were prepared which contained the 8 “b”sequences previously used in addition to the mixotope. Sera wereselected which previously scored positive on at least one of the eightdefined sequences as well as sera which scored negative. In total, 60sera were tested, of which 56 previously gave a positive reaction and 4were previously found to be negative. Of the 56 sera which hadpreviously scored positive, 21 reacted with only one or two of thepeptides on the strip or only gave a very weak reaction. (data notshown). The mixotope was recognized by approximately one-third of allthe sera tested. The reaction of some sera to the mixotope wassurprisingly strong, however, it may be possible that the collection ofE2/NSl sequences on which the mixotope was based is not trulyrepresentative. It is expected that the mixotope MAP will elicit theproduction of broad specificity antisera directed against theamino-terminus of E2/NS1.

EXAMPLE 23 Use of branched HCV N-terminal E2/NS1 region peptides forraising antibodies

Several sequences from the N-terminus of E2/NS1 were selected forsynthesis as multiple antigen peptides (MAP's) using the techniquedescribed by Tam (Proc. Natl. Acad. Sci. USA 85:5409-5413, 1988). Thestrategy used to synthesize the branched peptides is shown schematicallyin FIG. 16. Rabbits (two for each MAP) were given an initial injectionand were boosted once before blood was drawn for a first evaluation ofantibody production. The antisera were tested on LIA strips containing atotal of 16 E2 peptides (sequences derived from 8 type 1 isolates, “a”and “b” versions of each). Examination of the LIA strips reveals thatthere is considerable cross-reaction between the antibodies raised inthe rabbits and the various E2 peptides on the strips (FIG. 17). Thefact that both “a” and “b” variations can be found which are recognizedby the different antisera indicates that there is at least one epitopelocated in the region where these two versions overlap.

EXAMPLE 24 Diagnosis of HTLV infection using Biotinylated SyntheticPeptides

HTLV-I and II are antigenically related members of a family of oncogenicretroviruses. HTLV-I infection has been shown to be associated with twodisease syndromes: HTLV-I-associated myelopathy/tropical spasticparaparesis (neurological disorders) and adult T-cell leukemia (ATL). Incontrast, HTLV-II has not been conclusively linked to any known diseasesyndrome. This virus was originally isolated from a patient with hairycell leukemia, however, no causal relationship between HTLV-II infectionand the disease state could be established. Since HTLV-I infection hasdefinitely been demonstrated to have the potential to result in humandisease while HTLV-II infection has not, it is of clinical interest tobe able to differentiate between these two infectious agents. Sincethese two viruses are antigenically highly related, it is difficult todiscriminate between HTLV-I and HTLV-II infections when viral orrecombinant antigens are used for antibody detection. A number ofbiotinylated peptides were synthesized and evaluated for their abilityto detect antibodies raised in response to infection by either HTLV-I orHTLV-II. Some of the peptides were chosen because they contain epitopeswhich are highly conserved between HTLV-I and HTLV-II and shouldtherefore be useful reagents for detecting HTLV infection without regardto virus type. Still other peptides were chosen because they containepitopes which should allow HTLV-I and HTLV-II infections to bediscriminated. The peptides synthesized are as follows:

I-gp46-3:

Bio Gly Gly Val Leu Tyr Ser Pro Asn Val Ser Val Pro Ser Ser Ser Ser ThrLeu Leu Tyr Pro Ser Leu Ala

I-gp46-5:

Bio Gly Gly Tyr Thr Cys Ile Val Cys Ile Asp Arg Ala Ser Leu Ser Thr TrpHis Val Leu Tyr Ser Pro

I-gp46-4:

Bio Gly Gly Asn Ser Leu Ile Leu Pro Pro Phe Ser Leu Ser Pro Val Pro ThrLeu Gly Ser Arg Ser Arg Arg

I-gp46-6:

Bio Gly Gly Asp Ala Pro Gly Tyr Asp Pro Ile Trp Phe Leu Asn Thr Glu ProSer Gln Leu Pro Pro Thr Ala Pro Pro Leu Leu Pro His Ser Asn Leu Asp HisIle Leu Glu

I-p21-2:

Bio Gly Gly Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp Leu Leu Phe TrpGlu Gln Gly Gly Leu Cys Lys Ala Leu Gln Glu Gln Cys Arg Phe Pro

I-p19:

Bio Gly Gly Pro Pro Pro Pro Ser Ser Pro Thr His Asp Pro Pro Asp Ser AspPro Gln Ile Pro Pro Pro Tye Val Glu Pro Thr Ala Pro Gln Val Leu

II-gp52-1:

Bio Gly Gly Lys Lys Pro Asn Arg Gln Gly Leu Gly Tyr Tyr Ser Pro Ser TyrAsn Asp Pro

II-gp52-2:

Bio Gly Gly Asp Ala Pro Gly Tyr Asp Pro Leu Trp Phe Ile Thr Ser Glu ProThr Gln Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu HisVal Leu Thr

II-gp52-3:

Bio Gly Gly Tyr Ser Cys Met Val Cys Val Asp Arg Ser Ser Leu Ser Ser TrpHis Val Leu Tyr Thr Pro Asn Ile Ser Ile Pro Gln Gln Thr Ser Ser Arg ThrIle Leu Phe Pro Ser

II-p19:

Bio Gly Gly Pro Thr Thr Thr Pro Pro Pro Pro Pro Pro Pro Ser Pro Glu AlaHis Val Pro Pro Pro Tyr Val Glu Pro Thr Thr Thr Gln Cys Phe

A number of these peptides were used to prepare LIA strips for thedetection of antibodies to HTLV. Several of the peptides, such as I-p19and I-gp46-4, which are derived from regions of the HTLV-I p19 gagprotein and envelope glycoprotein, respectively, are expected to berecognized by antibodies produced as a result of both HTLV-I and HTLV-IIinfection since these sequences are highly homologous in the twoviruses. Others, such as I-gp46-3, I-gp46-4 for HTLV-I, and II-gp52-1,II-gp52-2 and II-gp52-3 for HTLV-II may be useful for detection ofantibodies as well as discrimination. Since there is some homologybetween the HTLV-I and HTLV-II sequences, cross-reactions are to beexpected. Nevertheless, the intensities of the reactions to the variouspeptides should reveal the identity of the virus to which the antibodieswere produced.

An example of LIA strips prepared with a number of the biotinylatedHTLV-I and HTLV-II peptides is shown in FIG. XXX. The LIA strips wereevaluated using a commercially available serum panel (Boston BiomedicaInc., mixed titer panel, PRP203). The test results are in completeagreement with the analysis provided by distributor. Only one sample(nr. 9) is positive for HTLV-I. Sample nr.12 is detected as positivebecause of the positive reaction to the peptide I-p19. This sample couldnot be differentiated using these peptides, nor could this sample bedifferentiated by any other test used by the distributor of the serumpanel. Sample nr. 11 was found to be negative and all other samples werefound to be positive for HTLV-II. In an additional experiment, an ELISAwas performed using all 10 of the biotinylated HTLV-I and HTLV-IIpeptides. The peptides were completed with streptavidin individually andthen mixed prior to coating. Some of the samples from the panel used toevaluated the LIA strips were used to evaluate the peptides in theELISA. These results are shown in table. The ELISA in this configurationcannot be used to differentiate HTLV-I and -II infections but shouldidentify HTLV-positive samples in general regardless of virus type. Theresults further demonstrate the utility of these peptides for thediagnosis of HTLV infection.

TABLE 1 Antibody recognition of biotinylated and unbiotinylated HIV-1and HIV-2 peptides TM- TM-HIV-1 HIV- Serum TM-HIV-1 Bio TM-HIV-2 2 BioHIV-1 0724 0.174 2.570 0.000 0.000 positive mm 0.051 2.579 0.000 0.000YEMO 0.162 2.357 0.000 0.000 PL 0.000 1.559 0.000 0.000 VE 0.052 2.5510.000 0.000 HIV-2 1400 0.000 0.000 0.000 1.982 positive AG 0.000 0.0000.000 2.323 53-3 0.000 0.000 0.000 2.365 Seronegative 194 0.000 0.0000.000 0.000 donors 195 0.000 0.000 0.000 0.000 180 0.000 0.005 0.0000.000 204 0.000 0.001 0.000 0.000

TABLE 2 Comparison of antibody recognition of biotinylated andunbiotinylated peptides from the V3 sequence of isolate HIV-1 mn. Sampleidentity V3-mn V3-mn Bio negative control 0.063 0.069 blank 0.053 0.051YS 1.442 2.784 DV 1.314 2.881 VE 1.717 overflow* OOST 6 1.025 2.855 OOST8 1.389 overflow* 3990 1.442 overflow* PL 0.531 2.351 MM 0.791 2.5424436 0.388 2.268 4438 0.736 2.554 266 0.951 2.591 OOST 4 1.106 overflow**Absorbance value greater than 3.000

TABLE 3 Comparison of antibody recognition of the biotinylated V3-mnpeptide bound to streptavidin and avidin Serum Streptavidin Avidin YS1.236 1.721 DV 1.041 1.748 PL 0.222 0.983 3990 1.391 1.854 VE 1.5261.908 4436 0.596 1.519 Control 0.050 0.063

TABLE 4 Comparison of antibody recognition of biotinylated andunbiotinylated HCV peptides. Table 4A Antibody binding to HCV peptide XISerum Unbiotinylated peptide XI Peptide XI 2 0.090 1.971 3 0.443 2.086 40.473 1.976 6 0.053 0.518 8 1.275 2.624 10 0.764 2.321 11 0.569 2.378 230.775 2.503 31 0.497 2.104 77 0.093 0.159 33 0.832 1.857 49 0.515 2.180negative serum 0.053 0.095

TABLE 4B Antibody binding to HCV peptide XVI Serum Unbiotinylatedpeptide XVI Peptide XVI 1 1.038 2.435 2 0.616 1.239 6 0.100 1.595 80.329 1.599 10 1.033 2.847 26 0.053 1.522 83 0.912 2.221 88 1.187 2.51989 0.495 1.530 91 0.197 2.169 95 0.109 1.484 99 0.814 2.045 100 0.4741.637 104 0.205 0.942 105 0.313 2.186 110 0.762 1.484 111 0.193 1.465112 0.253 1.084 113 0.833 2.535 116 0.058 1.918 120 0.964 2.332 114760.068 2.197 24758 0.071 0.062 266 0.712 2.262 8247 0.059 0.618 negativeserum 0.063 0.067

TABLE 4C Antibody binding to HCV peptide II Serum Unbiotinylated peptideII Peptide II 8241 0.444 0.545 8242 1.682 2.415 8243 2.181 2.306 82471.518 1.975 8250 0.110 0.357 8271 0.912 1.284 8273 2.468 2.769 82742.700 2.943 8275 1.489 2.030 8276 2.133 2.348 8277 1.771 2.572 82781.907 2.022 negative serum 0.047 0.070

TABLE 4D Antibody binding to HCV peptide III Serum Unbiotinylatedpeptide III Peptide III 8241 1.219 2.066 8242 1.976 2.197 8243 1.8592.368 8247 1.072 2.398 8248 2.742 2.918 8250 2.471 2.626 8271 1.4712.066 8272 2.471 2.638 8273 1.543 2.697 8274 2.503 2.905 8275 1.5952.640 8276 1.976 2.674 8277 0.735 2.327 negative serum 0.050 0.06

TABLE 4E Antibody binding to HCV peptide V Serum Unbiotinylated peptideV Peptide V 8272 0.589 1.220 8273 0.294 1.026 8274 1.820 2.662 82751.728 1.724 8276 2.194 2.616 8277 0.770 1.796 8278 1.391 1.746 82840.040 0.757 negative serum 0.047 0.070

TABLE 4F Antibody binding to HCV peptide IX Serum Unbiotinylated peptideIX Peptide IX 8315 2.614 2.672 8316 0.133 0.367 8317 0.855 1.634 83181.965 2.431 8320 0.721 0.896 8321 0.283 0.457 8326 2.219 2.540 negativeserum 0.052 0.005

TABLE 4G Antibody binding to HCV peptide XVIII Serum Unbiotinylatedpeptide XVIII Peptide XVIII 79 1.739 2.105 83 1.121 1.232 88 0.972 1.85889 2.079 2.309 91 2.202 2.132 99 1.253 1.526 104 1.864 1.998 105 1.5222.053 110 1.981 2.065 111 1.363 1.542 112 1.172 1.408 116 1.534 1.978120 1.599 2.031 1 2.523 2.691 33 1.463 1.813 39 0.068 0.213 47 2.1172.611 negative serum 0.001 0.001

TABLE 5 Peptide concentration* 3.0 1.0 0.3 0.1 0.03 0.01 coatingmethod** 1 2 1 2 1 2 1 2 1 2 1 2 Unbiotinylated HCV peptide II and HCVpeptide II sample positive 8320 2.718 2.278 2.684 2.163 2.684 2.0042.718 1.828 2.757 1.272 2.519 0.479 8242 1.427 0.539 1.368 0.408 1.3650.234 1.399 0.058 1.481 0.048 1.196 0.051 8243 1.668 1.341 1.652 1.2211.608 0.831 1.639 0.181 1.597 0.057 1.088 0.056 8318 2.016 0.791 1.9930.626 1.958 0.347 2.001 0.181 2.181 0.095 2.002 0.048 sample negative1747 0.064 0.049 0.071 0.046 0.046 0.041 0.045 0.044 0.045 0.043 0.0450.041 1781 0.057 0.053 0.055 0.053 0.051 0.045 0.047 0.046 0.049 0.0530.053 0.046 Unbiotinylated HCV peptide IX and HCV peptide IX samplepositive 8320 1.779 0.129 0.802 0.093 1.798 0.122 1.244 0.063 1.0070.057 0.461 0.059 8326 2.284 0.084 2.271 0.068 2.271 0.078 2.284 0.0682.193 0.051 1.812 0.049 8242 0.791 0.059 0.777 0.052 0.795 0.048 0.9110.046 0.496 0.047 0.215 0.049 8243 1.959 0.063 1.953 0.053 1.892 0.0511.834 0.051 1.421 0.051 0.639 0.054 sample negative 1747 0.051 0.0460.049 0.046 0.046 0.044 0.042 0.045 0.044 0.045 0.043 0.045 1781 0.0530.053 0.051 0.052 0.051 0.051 0.047 0.052 0.048 0.049 0.049 0.051Unbiotinylated HCV peptide XVIII and HCV peptide XVIII sample positive8326 2.315 0.052 2.331 0.053 2.331 0.053 2.331 0.049 2.219 0.051 1.8480.051 8242 0.749 0.053 0.839 0.049 0.873 0.048 0.946 0.047 1.188 0.0491.185 0.048 8243 0.671 0.057 0.627 0.053 0.629 0.054 0.661 0.051 0.6110.053 0.462 0.053 8318 2.391 0.051 2.396 0.045 2.392 0.047 2.409 0.0472.308 0.047 1.711 0.048 sample negative 1747 0.047 0.048 0.042 0.0450.061 0.046 0.044 0.045 0.058 0.044 0.042 0.047 1781 0.053 0.055 0.0480.054 0.048 0.051 0.048 0.051 0.051 0.051 0.045 0.053 *in micrograms permilliliter **1 biotinylated peptide on streptabidin coated plate 2unbiotinylated peptide coated directly

TABLE 6 Comparison of N- and C-terminally biotinylated TM-HIV-1 peptideTM-HIV-1 TM-HIV-1 Serum C-terminal biotin N-terminal biotin HIV positiveVE 2.079 2.240 OOST 6 1.992 2.003 MM 2.097 2.308 0724 2.322 2.291 DV0.903 1.579 PL 1.893 1.849 2049 1.780 2.058 3990 1.959 1.870 4438 1.6221.697 4436 2.190 2.110 OOST 7 1.728 2.027 OOST 8 2.117 2.237 OOST 92.119 2.222 VCM 2.131 2.263 1164 1.865 1.919 1252 2.244 2.356 0369/872.059 2.042 Seronegative blood 1784 0.000 0.000 donors 1747 0.000 0.0001733 0.014 0.000

TABLE 7 HCV peptide I HCV peptide I carboxy-biotinylated (coateddirectly) (bound to streptavidin-coated wells) HCV antibody- positivesera 8316 2.394 2.541 8318 2.385 2.404 8320 2.760 2.762 8326 0.525 1.7758329 2.633 2.672 8333 2.143 2.545 8334 2.271 2.549 8336 1.558 2.016 83441.878 2.010 8248 2.042 2.493 8244 0.077 1.399 8243 2.211 2.541 82421.367 2.389 8364 2.705 2.705 8374 1.070 2.151 8378 2.161 2.531 83301.985 2.651 8387 1.427 2.628 HCV antibody- negative sera F88 0.000 0.026F89 0.017 0.001 F76 0.000 0.022 F136 0.006 0.002 F8 0.000 0.000

TABLE 8 Use of mixtures of biotinylated peptides for antibody detectionMixture HCV peptide HCV peptide HCV peptide Mixture Mixture A MixtureTM-HIV-1- TM-HIV-2- V3-mn-BIO II-BIO IX-BIO XVIII-BIO A B Direct DirectSerum BIO Avidin BIO Avidin Avidin Avidin Avidin Avidin Avidin Avidincoating coating HCV 8243 0.108 0.109 0.114 1.430 1.213 0.118 1.590 1.6380.542 0.184 8247 0.042 0.048 0.052 1.356 0.756 0.046 0.840 1.149 0.0490.049 8248 0.043 0.046 0.048 2.287 0.047 0.905 1.859 2.154 0.407 0.0648269 0.053 0.049 0.056 1.213 0.051 1.513 0.923 1.268 0.078 0.067 82900.045 0.047 0.050 0.060 0.048 2.323 1.210 1.761 0.559 0.717 8278 0.0460.045 0.053 1.878 0.074 0.052 1.806 1.944 0.540 0.152 8273 0.053 0.0500.056 2.017 0.053 0.052 2.037 2.113 0.773 0.185 8285 0.134 0.163 0.1431.592 0.270 0.146 1.746 1.822 0.908 0.401 8291 0.048 0.050 0.053 1.5390.052 0.049 1.591 1.809 0.335 0.098 HIV-2 AG 0.054 2.065 0.068 0.0810.064 0.058 1.833 1.880 0.054 0.056 1400 0.051 1.781 0.055 0.121 0.0521.362 1.692 2.031 0.214 0.326 HIV-1 YS 0.046 0.046 2.201 0.048 0.0490.049 2.045 1.845 0.200 0.052 PL 1.974 0.051 1.321 0.052 0.056 0.0521.587 1.776 0.052 0.055 DV 1.329 0.048 2.340 0.047 0.049 0.047 1.9691.742 0.100 0.049 3990 1.602 0.054 2.319 0.054 0.066 0.056 2.217 1.9260.390 0.081 Blood donor 1785 0.046 0.047 0.048 0.045 0.050 0.047 0.0470.049 0.045 0.049 1794 0.124 0.090 0.091 0.153 0.098 0.104 0.152 0.1610.050 0.058 1784 0.044 0.046 0.046 0.045 0.050 0.047 0.047 0.047 0.0450.048 1782 0.052 0.057 0.059 0.057 0.062 0.053 0.057 0.059 0.049 0.056

TABLE 9 Sequences of the Core Epitopes of the HCV Core Protein HCV COREPROTEIN AMINO ACIDS 1-90 Positions of core epitopes (SEQ ID NO:) Epitope1A:

M S T I P K P Q R K T K R N T N R R P Q P Q R K T K R N T N R R P Q D VK F P G (453) (454) CORE 1 CORE 2 Epitope 1B:

M S T I P K P Q R K T K R N T N R R P Q P Q R K T K R N T N R R P Q D VK F P G (453) (454) CORE 1 CORE 2 Epitope 2:

P Q R K T K R N T N R R P Q D V K F P G R N T N R R P Q D V K F P G G GQ I V G (454) (455) CORE 2 CORE 3 Epitope 3A:

P G G G Q I V G G V Y L L P R R G P R L (456) CORE 5 Epitope 3B:

P G G G Q I V G G V Y L L P R R G P R L L P R R G P R L G V R A T R K TS E R S (456) (457) CORE 5 CORE 7 Epitope 3C:

L P R R G P R L G V R A T R K T S E R S (457) CORE 7 Epitope 4A:

T R K T S E R S Q P R G R R Q P I P K V (458) CORE 9 Epitope 4B:

T R K T S E R S Q P R G R R Q P I P K V R R Q P I P K V R R P E G R T WA Q P G (458) (459) CORE 9 CORE 11 Epitope 5A:

R R Q P I P K V R R P E G R T W A Q P G G R T W A Q P G Y P W P L Y G NE G C G (459) (600) CORE 11 CORE 13 Epitope 5B: (minor)

G R T W A Q P G Y P W P L Y G N E G C G (600) CORE 13

TABLE 10 Sequences of the Core Epitopes of the HCV NS4 Protein HCV NS4PROTEINS Positions of core epitopes (SEQ ID NO:) Epitope 1A:

L S G K P A I I P D R E V L Y R E F D E I I P D R E V L Y R E F D E M EE C S Q (460) (461) HCV1 HCV2 Epitope 2A:

V L Y R E F D E M E E C S Q H L P Y I E D E M E E C S Q H L P Y I E Q GM M L A S Q H L P Y I E Q G M M L A E Q F K Q K (462) (463) (464) HCV3HCV4 HCV5 Epitope 2B: (minor)

D E M E E C S Q H L P Y I E Q G M M L A S Q H L P Y I E Q G M M L A E QF K Q K (463) (464) HCV4 HCV5 Epitope 3A:

S Q H L P Y I E Q G M M L A E Q F K Q K I E Q G M M L A E Q F K Q K A LG L L Q L A E Q F K Q K A L G L L Q T A S R Q A (464) (465) (466) HCV5HCV6 HCV7 Epitope 3B:

I E Q G M M L A E Q F K Q K A L G L L Q L A E Q F K Q K A L G L L Q T AS R Q A (465) (466) HCV6 HCV7 Epitope 4:

L A E Q F K Q K A L G L L Q T A S R Q A Q K A L G L L Q T A S R Q A E VI A P A (466) (467) HCV7 HCV8

TABLE 11 Sequences of the Core Epitopes of the HCV NS5 Protein HCV NS5PROTEINS Positions of the core epitopes (SEQ ID NO:) Epitope 1A:

E D E R E I S V P A E I L R K S R R F A (471) NS5-25 Epitope 1B:

E D E R E I S V P A E I L R K S R R F A (471) NS5-25 Epitope 2:

L R K S R R F A Q A L P V W A R P D Y N (472) NS5-27 Epitope 3:

V W A R P D Y N P P L V E T W K K P D Y (473) NS5-29 Epitope 4: (minor)

V W A R D Y N P P L V E T W K K P D Y (473) NS5-29 Epitope 5:

E T W K K P D Y E P P V V H G C P L P P (474) NS5-31 Epitope 6:

E T W K K P D Y E P P V V H G C P L P P V H G C P L P P P K S P P V P PP R K K (474) (475) NS5-31 NS5-33

TABLE 12 Antibody binding of Various Core, NS4 and NS5 biotinylated20-mers by the 10 test sera PEPTID ELISA (O.D.) SERUM Core-2 Core-3Core-7 Core-9 HCV-2 HCV-5 HCV-7 NS5-25 NS5-27 NS5-31 8242 2.415 2.1970.632 2.315 2.114 1.625 1.252 0.268 2.318 2.453 8248 2.441 2.918 1.5292.021 0.142 0.182 1.963 0.054 0.388 1.511 8332 1.977 2.054 1.387 1.4550.392 0.575 0.945 0.047 2.130 2.290 8339 2.030 2.765 0.166 2.598 2.4970.043 0.041 1.495 2.359 2.757 8358 1.982 2.135 0.357 0.685 1.779 0.6230.598 0.069 2.249 0.182 8377 2.181 2.368 0.221 0.076 2.360 2.227 1.8291.092 2.336 1.378 8378 1.140 2.369 1.089 1.228 1.859 1.449 2.006 0.2791.602 2.337 8383 2.463 2.463 0.970 2.162 2.300 1.018 2.504 0.055 2.3901.378 8241 0.545 2.066 0.448 0.274 2.421 2.280 0.968 0.050 2.456 0.2738243 2.306 2.368 1.251 1.378 2.203 2.268 2.251 0.062 1.444 0.127

TABLE 13 Antibody recognition of individual E2/NS1 peptides. (percent ofall sera giving a positive reaction.) CL14-A  7 (51)  13.7% B 36 (51) 70.6% KATO-A  2 (51)  3.92% B 26 (51) 50.98% HCJ4-A 32 (51) 62.74% B 41(51) 80.39% FRENCH-A 30 (51)  58.8% B 42 (51) 82.35% YEK-A  7 (51)13.72% B 49 (51) 96.07% TAMI-A 12 (51) 23.52% B 36 (51) 70.58% 18CH1-A 5 (51)  9.8% B 30 (51) 58.82% CHIR-A 32 (51)  62.7% B 40 (51) 78.43%

TABLE 14 Overall Recognition of NB-Loop peptides. CON SC MN SF2 BH RFMAL ELI 70 Total SUM 287 261 275 258 108 146 140 24 6 COUNT 326 326 326326 326 326 326 326 326 gp120 positive % Reactive 88 80 84 79 33 45 43 72 Total SUM 287 261 275 258 108 146 140 24 6 COUNT 307 307 307 307 307307 307 307 307 HIV-V3 positive % Reactive 93 85 90 84 35 48 46 8 2

TABLE 15 Recognition of peptides according to geographical regionEUROPEAN % AFRICAN % BRAZILIAN % Consensus 98 Consensus 89 Consensus 82HIV-1 (SC) 98 HIV-1 (MN) 85 HIV-1 (MN) 78 HIV-1 (SF2) 98 HIV-1 (SF2) 79HIV-1 (SC) 75 HIV-1 (MN) 97 HIV-1 (SC) 73 HIV-1 (SF2) 72 HIV-1 (RF) 75HIV-1 (MAL) 60 HIV-1 (RF) 38 HIV-1 (MAL) 68 HIV-1 (RF) 34 HIV-1 (MAL) 30HIV-1 (IIIB) 61 HIV-1 (IIIB) 27 HIV-1 (IIIB) 26 HIV-1 (ELI) 8 HIV-1(ELI) 13 HIV-1 (ELI) 5 ANT 70 2 ANT 70 2 ANT 70 2

TABLE 16 Recognition of European, African and Brazilian HIV-1antibody-positive sera to HIV-1 V3 loop peptides V3-con and V3-368V3-con V3-368 V3con-V3-368 European sera number tested 36 36 36 numberpositive 33 4 33 number negative 0 12 0 number borderline 3 20 3 percentpositive 92 11 92 percent negative 0 33 0 percent borderline 8 56 8African sera number tested 45 45 45 number positive 40 5 40 numbernegative 4 31 2 number borderline 1 9 3 percent positive 89 11 89percent negative 9 69 4 percent borderline 2 20 7 Brazilian sera numbertested 36 36 36 number positive 30 16 35 number negative 1 5 1 numberborderline 5 15 0 percent positive 83.3 44.4 97.2 percent negative 2.813.9 2.8 percent borderline 13.9 41.7 0

TABLE 17 Recognition of HIV-2 positive sera to peptides from the V2 loopregion of HIV-2 V3-GB12 V3-239 number tested 21 21 number positive 21 19number negative 0 0 number borderline 0 2 percent positive 100 90.5percent negative 0 0 percent borderline 0 9.5

TABLE 18 Antibody recognition of hybrid peptides A. Ser- NS4 NS5 CoreLIA Discrep- um Epitope 1 Epitope 5 Epitope 2 Epi-152 ancies 5241 — — ——weak 5242 — — — — 5243 — — — — 5248 — — — — 5332 — — — — 5339 — — — —5358 — — — — 5377 — — — — 5378 — — — — 5383 — — — — B. Ser- NS5 NS4 CoreEpi- LIA Discrep- um Epitope 3 Epitope 3B tope 3A Epi-33B3A ancies 6241— — — — — 6242 — — — — 6243 — — — — 6248 — — — — 6332 — — — — — 6339 — —— — 6358 — — — — 6377 — — — — 6378 — — — — 6383 — — — — C. Ser- Core NS4NS5 LIA Discrep- um Epitope 4B Epitope 2A Epitope 5 Epi-4B2A5 ancies8241 — — — — 8242 — — — — 8243 — — — — — — — — 8248 — — — — 8332 — — — ——weak 8339 — — — — 8358 — — — — — ? 8377 — — — — — 8378 — — — — 8383 — —— —

TABLE 19 ANTIBODY RECOGNITION OF HTLV PEPTIDES Serum number Opticaldensity 1 0.303 2 3.001 3 0.644 4 1.262 6 3.001 7 2.623 9 2.607 (HTLV-1)10 3.001 11 0.058 (negative) 13 3.001 14 3.001 15 0.850 16 0.278 191.048 20 3.001 21 0.805 22 0.812 23 3.001 24 0.405 25 1.521

600 1 9 PRT Human immunodeficiency virus 1 Ile Trp Gly Cys Ser Gly LysIle Cys 1 5 2 18 PRT Human immunodeficiency virus 2 Ile Trp Gly Cys SerGly Lys Leu Ile Cys Thr Thr Ala Val Pro Asn 1 5 10 15 Ala Ser 3 19 PRTHuman immunodeficiency virus 3 Glu Arg Tyr Leu Lys Asp Gln Gln Leu LeuGly Ile Trp Gly Cys Gly 1 5 10 15 Lys Leu Ile 4 16 PRT Humanimmunodeficiency virus 4 Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr LeuLys Asp Gln Leu 1 5 10 15 5 10 PRT Human immunodeficiency virus 5 LeuTrp Gly Cys Lys Gly Lys Leu Val Cys 1 5 10 6 22 PRT Humanimmunodeficiency virus 6 Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser GlyLys His Ile Thr 1 5 10 15 Thr Asn Val Pro Trp Asn 20 7 22 PRT Humanimmunodeficiency virus 7 Asn Asn Thr Arg Lys Ser Ile His Ile Gly Pro GlyArg Ala Phe Thr 1 5 10 15 Thr Gly Glu Ile Ile Gly 20 8 34 PRT Humanimmunodeficiency virus 8 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser IleHis Ile Gly Gly 1 5 10 15 Arg Ala Phe Tyr Thr Thr Gly Glu Ile Ile GlyAsp Ile Arg Gln Ala 20 25 30 His Cys 9 22 PRT Human immunodeficiencyvirus 9 Asn Asn Thr Arg Lys Ser Ile Tyr Ile Gly Pro Gly Arg Ala Phe Thr1 5 10 15 Thr Gly Arg Ile Ile Gly 20 10 22 PRT Human immunodeficiencyvirus 10 Asn Asn Thr Thr Arg Ser Ile His Ile Gly Pro Gly Arg Ala Phe Ala1 5 10 15 Thr Gly Asp Ile Ile Gly 20 11 22 PRT Human immunodeficiencyvirus 11 Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Thr1 5 10 15 Thr Lys Asn Ile Ile Gly 20 12 23 PRT Human immunodeficiencyvirus 12 Asn Asn Thr Arg Lys Ser Ile Thr Lys Gly Pro Gly Arg Val Ile Tyr1 5 10 15 Ala Thr Gly Gln Ile Ile Gly 20 13 22 PRT Humanimmunodeficiency virus 13 Asn Asn Thr Arg Arg Gly Ile His Phe Gly ProGly Gln Ala Leu Tyr 1 5 10 15 Thr Thr Gly Ile Val Gly 20 14 24 PRT Humanimmunodeficiency virus 14 Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln ArgGly Pro Gly Arg Ala 1 5 10 15 Phe Val Thr Ile Gly Lys Ile Gly 20 15 22PRT Human immunodeficiency virus 15 Gln Asn Thr Arg Gln Arg Thr Pro IleGly Leu Gly Gln Ser Leu Tyr 1 5 10 15 Thr Thr Arg Ser Arg Ser 20 16 21PRT Human immunodeficiency virus 16 Gln Ile Asp Ile Gln Glu Met Arg IleGly Pro Met Ala Trp Tyr Ser 1 5 10 15 Met Gly Ile Gly Gly 20 17 23 PRTHuman immunodeficiency virus 17 Asn Asn Thr Arg Arg Gly Ile His Met GlyTrp Gly Arg Thr Phe Tyr 1 5 10 15 Ala Thr Gly Glu Ile Ile Gly 20 18 36PRT Human immunodeficiency virus 18 Arg Asp Asn Trp Arg Ser Glu Leu TyrLys Tyr Lys Val Val Lys Ile 1 5 10 15 Glu Pro Leu Gly Val Ala Pro ThrLys Ala Lys Arg Arg Val Val Gln 20 25 30 Arg Glu Lys Arg 35 19 10 PRTHuman immunodeficiency virus 19 Ser Trp Gly Cys Ala Phe Arg Gln Val Cys1 5 10 20 20 PRT Human immunodeficiency virus 20 Lys Tyr Leu Gln Asp GlnAla Arg Leu Asn Ser Trp Gly Cys Ala Phe 1 5 10 15 Arg Gln Val Cys 20 2123 PRT Human immunodeficiency virus 21 Asn Lys Thr Val Leu Pro Ile ThrPhe Met Ser Gly Phe Lys Phe His 1 5 10 15 Ser Gln Pro Val Ile Asn Lys 2022 22 PRT Human immunodeficiency virus 22 Asn Lys Thr Val Val Pro IleThr Leu Met Ser Gly Leu Val Phe His 1 5 10 15 Ser Gln Pro Ile Asn Lys 2023 22 PRT Human immunodeficiency virus 23 Asn Lys Thr Val Leu Pro ValThr Ile Met Ser Gly Leu Val Phe His 1 5 10 15 Ser Gln Pro Ile Asn Asp 2024 10 PRT Chimpanzee Immunodeficiency virus 24 Leu Trp Gly Cys Ser GlyLys Ala Val Cys 1 5 10 25 10 PRT Simian immunodeficiency virus 25 SerTrp Gly Cys Ala Trp Lys Gln Val Cys 1 5 10 26 10 PRT Simianimmunodeficiency virus 26 Gln Trp Gly Cys Ser Trp Ala Gln Val Cys 1 5 1027 22 PRT Human T-cell lymphotropic virus 27 Val Leu Tyr Ser Pro Asn ValSer Val Pro Ser Ser Ser Ser Thr Leu 1 5 10 15 Leu Tyr Pro Ser Leu Ala 2028 21 PRT Human T-cell lymphotropic virus 28 Tyr Thr Cys Ile Val Cys IleAsp Arg Ala Ser Leu Ser Thr Trp His 1 5 10 15 Val Leu Tyr Ser Pro 20 2922 PRT Human T-cell lymphotropic virus 29 Asn Ser Leu Ile Leu Pro ProPhe Ser Leu Ser Pro Val Pro Thr Leu 1 5 10 15 Gly Ser Arg Ser Arg Arg 2030 36 PRT Human T-cell lymphotropic virus 30 Asp Ala Pro Gly Tyr Asp ProIle Trp Phe Leu Asn Thr Glu Pro Ser 1 5 10 15 Gln Leu Pro Pro Thr AlaPro Pro Leu Leu Pro His Ser Asn Leu Asp 20 25 30 His Ile Leu Glu 35 3131 PRT Human T-cell lymphotropic virus 31 Gln Tyr Ala Ala Gln Asn ArgArg Gly Leu Asp Leu Leu Phe Trp Glu 1 5 10 15 Gln Gly Gly Leu Cys LysAla Leu Gln Glu Gln Cys Arg Phe Pro 20 25 30 32 31 PRT Human T-celllymphotropic virus 32 Pro Pro Pro Pro Ser Ser Pro Thr His Asp Pro ProAsp Ser Asp Pro 1 5 10 15 Gln Ile Pro Pro Pro Tyr Val Glu Pro Thr AlaPro Gln Val Leu 20 25 30 33 18 PRT Human T-cell lymphotropic virus 33Lys Lys Pro Asn Arg Gln Gly Leu Gly Tyr Tyr Ser Pro Ser Tyr Asn 1 5 1015 Asp Pro 34 36 PRT Human T-cell lymphotropic virus 34 Asp Ala Pro GlyTyr Asp Pro Leu Trp Phe Ile Thr Ser Glu Pro Thr 1 5 10 15 Gln Pro ProPro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu 20 25 30 His Val LeuThr 35 35 38 PRT Human T-cell lymphotropic virus 35 Tyr Ser Cys Met ValCys Val Asp Arg Ser Ser Leu Ser Ser Trp His 1 5 10 15 Val Leu Tyr ThrPro Asn Ile Ser Ile Pro Gln Gln Thr Ser Ser Arg 20 25 30 Thr Ile Leu PhePro Ser 35 36 30 PRT Human T-cell lymphotropic virus 36 Pro Thr Thr ThrPro Pro Pro Pro Pro Pro Pro Ser Pro Glu Ala His 1 5 10 15 Val Pro ProPro Tyr Val Glu Pro Thr Thr Thr Gln Cys Phe 20 25 30 37 20 PRT HepatitisC virus 37 Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn ThrAsn 1 5 10 15 Arg Arg Pro Gln 20 38 20 PRT Hepatitis C virus 38 Pro GlnArg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val 1 5 10 15 LysPhe Pro Gly 20 39 11 PRT Hepatitis C virus 39 Gln Arg Lys Thr Lys ArgAsn Thr Asn Arg Arg 1 5 10 40 20 PRT Hepatitis C virus 40 Arg Asn ThrAsn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly 1 5 10 15 Gln IleVal Gly 20 41 20 PRT Hepatitis C virus 41 Leu Pro Arg Arg Gly Pro ArgLeu Gly Val Arg Ala Thr Arg Lys Thr 1 5 10 15 Ser Glu Arg Ser 20 42 20PRT Hepatitis C virus 42 Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly ProArg Leu Gly Val 1 5 10 15 Arg Ala Thr Arg 20 43 20 PRT Hepatitis C virus43 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 1 510 15 Ile Pro Lys Val 20 44 20 PRT Hepatitis C virus 44 Arg Ser Gln ProArg Gly Arg Arg Gln Pro Ile Pro Lys Val Arg Arg 1 5 10 15 Pro Glu GlyArg 20 45 20 PRT Hepatitis C virus 45 Arg Arg Gln Pro Ile Pro Lys ValArg Arg Pro Glu Gly Arg Thr Trp 1 5 10 15 Ala Gln Pro Gly 20 46 20 PRTHepatitis C virus 46 Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro LeuTyr Gly Asn 1 5 10 15 Glu Gly Cys Gly 20 47 30 PRT Hepatitis C virus 47Met Ser Thr Ile Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 1 5 1015 Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 4840 PRT Hepatitis C virus 48 Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly ProArg Leu Gly Val Arg 1 5 10 15 Arg Ala Thr Arg Lys Thr Ser Glu Arg SerGln Pro Arg Gly Arg Arg 20 25 30 Gln Pro Ile Pro Lys Val Arg Arg 35 4049 20 PRT Hepatitis C virus 49 Leu Ser Gly Lys Pro Ala Ile Ile Pro AspArg Glu Val Leu Tyr Arg 1 5 10 15 Glu Phe Asp Glu 20 50 20 PRT HepatitisC virus 50 Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu MetGlu 1 5 10 15 Glu Cys Ser Gln 20 51 20 PRT Hepatitis C virus 51 Val LeuTyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu 1 5 10 15 ProTyr Ile Glu 20 52 20 PRT Hepatitis C virus 52 Asp Glu Met Glu Glu CysSer Gln His Leu Pro Tyr Ile Glu Gln Gly 1 5 10 15 Met Met Leu Ala 20 5320 PRT Hepatitis C virus 53 Ser Gln His Leu Pro Tyr Ile Glu Gln Gly MetMet Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys 20 54 20 PRT Hepatitis Cvirus 54 Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu1 5 10 15 Gly Leu Leu Gln 20 55 20 PRT Hepatitis C virus 55 Leu Ala GluGln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala 1 5 10 15 Ser ArgGln Ala 20 56 20 PRT Hepatitis C virus 56 Gln Lys Ala Leu Gly Leu LeuGln Thr Ala Ser Arg Gln Ala Glu Val 1 5 10 15 Ile Ala Pro Ala 20 57 31PRT Hepatitis C virus 57 Ser Gln His Leu Pro Tyr Ile Glu Gln Glu Met LeuAla Glu Gln Phe 1 5 10 15 Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr AlaSer Arg Gln Ala 20 25 30 58 20 PRT Hepatitis C virus 58 Gly Glu Gly AlaVal Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser 1 5 10 15 Arg Gly AsnHis 20 59 20 PRT Hepatitis C virus 59 Glu Asp Glu Arg Glu Ile Ser ValPro Ala Glu Ile Leu Arg Lys Ser 1 5 10 15 Arg Arg Phe Ala 20 60 20 PRTHepatitis C virus 60 Leu Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro ValTrp Ala Arg 1 5 10 15 Pro Asp Tyr Asn 20 61 20 PRT Hepatitis C virus 61Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys 1 5 1015 Lys Pro Asp Tyr 20 62 20 PRT Hepatitis C virus 62 Glu Thr Trp Lys LysPro Asp Tyr Glu Pro Pro Val Val His Gly Cys 1 5 10 15 Pro Leu Pro Pro 2063 20 PRT Hepatitis C virus 63 Val His Gly Cys Pro Leu Pro Pro Pro LysSer Pro Pro Val Pro Pro 1 5 10 15 Pro Arg Lys Lys 20 64 37 PRT HepatitisC virus 64 Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg LysSer 1 5 10 15 Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp AlaArg Pro 20 25 30 Asp Tyr Asp Tyr Asn 35 65 34 PRT Hepatitis C virus 65Gly Glu Thr Tyr Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser Thr 1 5 1015 Leu Ala Ser Leu Phe Ser Pro Gly Ala Ser Gln Arg Ile Gln Leu Val 20 2530 Asn Thr 66 22 PRT Hepatitis C virus 66 Gly Glu Thr Tyr Thr Ser GlyGly Ala Ala Ser His Thr Thr Ser Thr 1 5 10 15 Leu Ala Ser Leu Phe Ser 2067 24 PRT Hepatitis C virus 67 Ser His Thr Thr Ser Thr Leu Ala Ser LeuPhe Ser Pro Gly Ala Ser 1 5 10 15 Gln Arg Ile Gln Leu Val Asn Thr 20 6834 PRT Hepatitis C virus 68 Gly His Thr Arg Val Ser Gly Gly Ala Ala AlaSer Asp Thr Arg Gly 1 5 10 15 Leu Val Ser Leu Phe Ser Pro Gly Ser AlaGln Lys Ile Gln Leu Val 20 25 30 Asn Thr 69 22 PRT Hepatitis C virus 69Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly 1 5 1015 Leu Val Ser Leu Phe Ser 20 70 24 PRT Hepatitis C virus 70 Ala Ser AspThr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala 1 5 10 15 Gln LysIle Gln Leu Val Asn Thr 20 71 34 PRT Hepatitis C virus 71 Gly His ThrArg Val Thr Gly Gly Val Gln Gly His Val Thr Cys Thr 1 5 10 15 Leu ThrSer Leu Phe Arg Pro Gly Ala Ser Gln Lys Ile Gln Leu Val 20 25 30 Asn Thr72 22 PRT Hepatitis C virus 72 Gly His Thr Arg Val Thr Gly Gly Val GlnGly His Val Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu Phe Arg 20 73 24 PRTHepatitis C virus 73 Gly His Val Thr Cys Thr Leu Thr Ser Leu Phe Arg ProGly Ala Ser 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 74 34 PRTHepatitis C virus 74 Gly His Thr His Val Thr Gly Gly Arg Val Ala Ser SerThr Gln Ser 1 5 10 15 Leu Val Ser Trp Leu Ser Gln Gly Pro Ser Gln LysIle Gln Leu Val 20 25 30 Asn Thr 75 22 PRT Hepatitis C virus 75 Gly HisThr His Val Thr Gly Gly Arg Val Ala Ser Ser Thr Gln Ser 1 5 10 15 LeuVal Ser Trp Leu Ser 20 76 24 PRT Hepatitis C virus 76 Ala Ser Ser ThrGln Ser Leu Val Ser Trp Leu Ser Gln Gly Pro Ser 1 5 10 15 Gln Lys IleGln Leu Val Asn Thr 20 77 34 PRT Hepatitis C virus 77 Gly Asp Thr HisVal Thr Gly Gly Ala Gln Ala Lys Thr Thr Asn Arg 1 5 10 15 Leu Val SerMet Phe Ala Ser Gly Pro Ser Gln Lys Ile Gln Leu Ile 20 25 30 Asn Thr 7822 PRT Hepatitis C virus 78 Gly Asp Thr His Val Thr Gly Gly Ala Gln AlaLys Thr Thr Asn Arg 1 5 10 15 Leu Val Ser Met Phe Ala 20 79 24 PRTHepatitis C virus 79 Ala Lys Thr Thr Asn Arg Leu Val Ser Met Phe Ala SerGly Pro Ser 1 5 10 15 Gln Lys Ile Gln Leu Ile Asn Thr 20 80 34 PRTHepatitis C virus 80 Ala Glu Thr Tyr Thr Ser Gly Gly Asn Ala Gly His ThrMet Thr Gly 1 5 10 15 Ile Val Arg Phe Phe Ala Pro Gly Pro Lys Gln AsnVal His Leu Ile 20 25 30 Asn Thr 81 22 PRT Hepatitis C virus 81 Ala GluThr Tyr Thr Ser Gly Gly Asn Ala Gly His Thr Met Thr Gly 1 5 10 15 IleVal Arg Phe Phe Ala 20 82 24 PRT Hepatitis C virus 82 Gly His Thr MetThr Gly Ile Val Arg Phe Phe Ala Pro Gly Pro Lys 1 5 10 15 Gln Asn ValHis Leu Ile Asn Thr 20 83 34 PRT Hepatitis C virus 83 Ala Glu Thr IleVal Ser Gly Gly Gln Ala Ala Arg Ala Met Ser Gly 1 5 10 15 Leu Val SerLeu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile 20 25 30 Asn Thr 8422 PRT Hepatitis C virus 84 Ala Glu Thr Ile Val Ser Gly Gly Gln Ala AlaArg Ala Met Ser Gly 1 5 10 15 Leu Val Ser Leu Phe Thr 20 85 24 PRTHepatitis C virus 85 Ala Arg Ala Met Ser Gly Leu Val Ser Leu Phe Thr ProGly Ala Lys 1 5 10 15 Gln Asn Ile Gln Leu Ile Asn Thr 20 86 34 PRTHepatitis C virus 86 Ala Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg ThrThr Gln Gly 1 5 10 15 Leu Val Ser Leu Phe Ser Arg Gly Ala Lys Gln AspIle Gln Leu Ile 20 25 30 Asn Thr 87 22 PRT Hepatitis C virus 87 Ala GluThr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gln Gly 1 5 10 15 LeuVal Ser Leu Phe Ser 20 88 24 PRT Hepatitis C virus 88 Ala Arg Thr ThrGln Gly Leu Val Ser Leu Phe Ser Arg Gly Ala Lys 1 5 10 15 Gln Asp IleGln Leu Ile Asn Thr 20 89 34 PRT Hepatitis C virus 89 Ala Gln Thr HisThr Val Gly Gly Ser Thr Ala His Asn Ala Arg Thr 1 5 10 15 Leu Thr GlyMet Phe Ser Leu Gly Ala Arg Gln Lys Ile Gln Leu Ile 20 25 30 Asn Thr 9022 PRT Hepatitis C virus 90 Ala Gln Thr His Thr Val Gly Gly Ser Thr AlaHis Asn Ala Arg Thr 1 5 10 15 Leu Thr Gly Met Phe Ser 20 91 24 PRTHepatitis C virus 91 Ala His Asn Ala Arg Thr Leu Thr Gly Met Phe Ser LeuGly Ala Arg 1 5 10 15 Gln Lys Ile Gln Leu Ile Asn Thr 20 92 20 PRTHepatitis C virus 92 Val Asn Gln Arg Ala Val Val Ala Pro Asp Lys Glu ValLeu Tyr Glu 1 5 10 15 Ala Phe Asp Glu 20 93 20 PRT Hepatitis C virus 93Val Ala Pro Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu 1 5 1015 Glu Cys Ala Ser 20 94 20 PRT Hepatitis C virus 94 Asp Glu Met Glu GluCys Ala Ser Arg Ala Ala Leu Ile Glu Glu Gly 1 5 10 15 Gln Arg Ile Ala 2095 20 PRT Hepatitis C virus 95 Ala Ser Arg Ala Ala Leu Ile Glu Glu GlyGln Arg Ile Ala Glu Met 1 5 10 15 Leu Lys Ser Lys 20 96 20 PRT HepatitisC virus 96 Ile Glu Glu Gly Gln Arg Ile Ala Glu Met Leu Lys Ser Lys IleGln 1 5 10 15 Gly Leu Leu Gln 20 97 20 PRT Hepatitis C virus 97 Ile AlaGlu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala 1 5 10 15 SerLys Gln Ala 20 98 20 PRT Hepatitis C virus 98 Ser Lys Ile Gln Gly LeuLeu Gln Gln Ala Ser Lys Gln Ala Gln Asp 1 5 10 15 Ile Gln Pro Ala 20 9920 PRT Hepatitis C virus 99 Arg Ser Asp Leu Glu Pro Ser Ile Pro Ser GluTyr Met Leu Pro Lys 1 5 10 15 Lys Arg Phe Pro 20 100 20 PRT Hepatitis Cvirus 100 Met Leu Pro Lys Lys Arg Phe Pro Pro Ala Leu Pro Ala Trp AlaArg 1 5 10 15 Pro Asp Tyr Asn 20 101 20 PRT Hepatitis C virus 101 AlaTrp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Ser Trp Lys 1 5 10 15Arg Pro Asp Tyr 20 102 20 PRT Hepatitis C virus 102 Glu Ser Trp Lys ArgPro Asp Tyr Gln Pro Ala Thr Val Ala Gly Cys 1 5 10 15 Ala Leu Pro Pro 20103 20 PRT Hepatitis C virus 103 Val Ala Gly Cys Ala Leu Pro Pro Pro LysLys Thr Pro Thr Pro Pro 1 5 10 15 Pro Arg Arg Arg 20 104 20 PRTHepatitis C virus 104 Leu Gly Gly Lys Pro Ala Ile Val Pro Asp Lys GluVal Leu Tyr Gln 1 5 10 15 Gln Tyr Asp Glu 20 105 20 PRT Hepatitis Cvirus 105 Ser Gln Ala Ala Pro Tyr Ile Glu Gln Ala Gln Val Ile Ala HisGln 1 5 10 15 Phe Lys Glu Lys 20 106 22 PRT Hepatitis C virus 106 IleAla His Gln His Gln Phe Lys Glu Lys Val Leu Gly Leu Leu Gln 1 5 10 15Arg Ala Thr Gln Gln Gln 20 107 31 PRT Hepatitis C virus 107 Ile Pro AspArg Glu Val Leu Tyr Arg Gly Gly Lys Lys Pro Asp Tyr 1 5 10 15 Glu ProPro Val Gly Gly Arg Arg Pro Gln Asp Val Lys Phe Pro 20 25 30 108 31 PRTHepatitis C virus 108 Trp Ala Arg Pro Asp Tyr Asn Pro Pro Gly Gly GlnPhe Lys Gln Lys 1 5 10 15 Ala Leu Gly Leu Gly Ser Gly Val Tyr Leu LeuPro Arg Arg Gly 20 25 30 109 31 PRT Hepatitis C virus 109 Arg Gly ArgArg Gln Pro Ile Pro Lys Gly Gly Ser Gln His Leu Pro 1 5 10 15 Tyr IleGlu Gln Ser Gly Pro Val Val His Gly Cys Pro Leu Pro 20 25 30 110 10 PRTHuman immunodeficiency virus 110 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys1 5 10 111 13 PRT Human immunodeficiency virus 111 Gly Gly Gly Ile TrpGly Cys Ser Gly Lys Leu Ile Cys 1 5 10 112 10 PRT Human immunodeficiencyvirus 112 Ser Trp Gly Cys Ala Phe Arg Gln Val Cys 1 5 10 113 13 PRTHuman immunodeficiency virus 113 Gly Gly Gly Ser Trp Gly Cys Ala Phe ArgGln Val Cys 1 5 10 114 23 PRT Human immunodeficiency virus 114 Tyr AsnLys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr 1 5 10 15 ThrThr Lys Asn Ile Ile Gly 20 115 24 PRT Human immunodeficiency virus 115Gly Gly Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala 1 5 1015 Phe Thr Thr Lys Asn Ile Ile Gly 20 116 20 PRT Hepatitis C virus 116Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln 1 5 1015 Phe Lys Gln Lys 20 117 20 PRT Hepatitis C virus 117 Leu Arg Lys SerArg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg 1 5 10 15 Pro Asp TyrAsn 20 118 19 PRT Hepatitis C virus 118 Pro Gln Arg Lys Thr Lys Arg AsnThr Asn Arg Arg Pro Gln Asp Val 1 5 10 15 Lys Phe Gly 119 20 PRTHepatitis C virus 119 Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys PhePro Gly Gly Gly 1 5 10 15 Gln Ile Val Gly 20 120 20 PRT Hepatitis Cvirus 120 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg GlnPro 1 5 10 15 Ile Pro Lys Val 20 121 20 PRT Hepatitis C virus 121 IleIle Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu 1 5 10 15Glu Cys Ser Gln 20 122 20 PRT Hepatitis C virus 122 Glu Thr Trp Lys LysPro Asp Tyr Glu Pro Pro Val Val His Gly Cys 1 5 10 15 Pro Leu Pro Pro 20123 19 PRT Hepatitis C virus 123 Met Ser Thr Ile Pro Lys Pro Gln Arg LysThr Lys Arg Asn Thr Asn 1 5 10 15 Arg Pro Gln 124 21 PRT Hepatitis Cvirus 124 Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn ThrAsn 1 5 10 15 Arg Pro Gln Gly Gly 20 125 34 PRT Hepatitis C virus 125Gly Glu Thr Tyr Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser Thr 1 5 1015 Leu Ala Ser Leu Phe Ser Pro Gly Ala Ser Gln Arg Ile Gln Leu Val 20 2530 Asn Thr 126 34 PRT Hepatitis C virus 126 Gly His Thr Arg Val Ser GlyGly Ala Ala Ala Ser Asp Thr Arg Gly 1 5 10 15 Leu Val Ser Leu Phe SerPro Gly Ser Ala Gln Lys Ile Gln Leu Val 20 25 30 Asn Thr 127 34 PRTHepatitis C virus 127 Gly His Thr Arg Val Thr Gly Gly Val Gln Gly HisVal Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu Phe Arg Pro Gly Ala Ser GlnLys Ile Gln Leu Val 20 25 30 Asn Thr 128 34 PRT Hepatitis C virus 128Gly His Thr His Val Thr Gly Gly Arg Val Ala Ser Ser Thr Gln Ser 1 5 1015 Leu Val Ser Trp Leu Ser Gln Gly Pro Ser Gln Lys Ile Gln Leu Val 20 2530 Asn Thr 129 34 PRT Hepatitis C virus 129 Gly Asp Thr His Val Thr GlyGly Ala Gln Ala Lys Thr Thr Asn Arg 1 5 10 15 Leu Val Ser Met Phe AlaSer Gly Pro Ser Gln Lys Ile Gln Leu Ile 20 25 30 Asn Thr 130 34 PRTHepatitis C virus 130 Ala Glu Thr Tyr Thr Ser Gly Gly Asn Ala Gly HisThr Met Thr Gly 1 5 10 15 Ile Val Arg Phe Phe Ala Pro Gly Pro Lys GlnAsn Val His Leu Ile 20 25 30 Asn Thr 131 34 PRT Hepatitis C virus 131Ala Glu Thr Ile Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser Gly 1 5 1015 Leu Val Ser Leu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile 20 2530 Asn Thr 132 34 PRT Hepatitis C virus 132 Ala Glu Thr Tyr Thr Thr GlyGly Ser Thr Ala Arg Thr Thr Gln Gly 1 5 10 15 Leu Val Ser Leu Phe SerArg Gly Ala Lys Gln Asp Ile Gln Leu Ile 20 25 30 Asn Thr 133 20 PRTHepatitis C virus 133 Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr LysArg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln 20 134 15 PRT Hepatitis Cvirus 134 Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro 15 10 15 135 20 PRT Hepatitis C virus 135 Arg Asn Thr Asn Arg Arg Pro GlnAsp Val Lys Phe Pro Gly Gly Gly 1 5 10 15 Gln Ile Val Gly 20 136 32 PRTHepatitis C virus 136 Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr LysArg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly GlyGly Gln Ile Val Gly 20 25 30 137 20 PRT Hepatitis C virus 137 Val GlyGly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 1 5 10 15 ArgAla Thr Arg 20 138 20 PRT Hepatitis C virus 138 Leu Pro Arg Arg Gly ProArg Leu Gly Val Arg Ala Thr Arg Lys Thr 1 5 10 15 Ser Glu Arg Ser 20 13920 PRT Hepatitis C virus 139 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro ArgGly Arg Arg Gln Pro 1 5 10 15 Ile Pro Glu Val 20 140 20 PRT Hepatitis Cvirus 140 Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Glu Val ArgArg 1 5 10 15 Pro Glu Gly Arg 20 141 39 PRT Hepatitis C virus 141 GlyGly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg 1 5 10 15Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln 20 25 30Pro Ile Pro Glu Val Arg Arg 35 142 20 PRT Hepatitis C virus 142 Ser GlnHis Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln 1 5 10 15 PheLys Gln Lys 20 143 20 PRT Hepatitis C virus 143 Leu Ala Glu Gln Phe LysGln Lys Ala Leu Gly Leu Leu Gln Thr Ala 1 5 10 15 Ser Arg Gln Ala 20 14432 PRT Hepatitis C virus 144 Ser Gln His Leu Pro Tyr Ile Glu Gln Gly MetMet Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys Ala Leu Gly Leu Leu GlnThr Ala Ser Arg Gln Ala 20 25 30 145 20 PRT Hepatitis C virus 145 GluAsp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser 1 5 10 15Arg Arg Phe Ala 20 146 20 PRT Hepatitis C virus 146 Leu Arg Lys Ser ArgArg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg 1 5 10 15 Pro Asp Tyr Asn 20147 32 PRT Hepatitis C virus 147 Glu Asp Glu Arg Glu Ile Ser Val Pro AlaGlu Ile Leu Arg Lys Ser 1 5 10 15 Arg Arg Phe Ala Gln Ala Leu Pro ValTrp Ala Arg Pro Asp Tyr Asn 20 25 30 148 20 PRT Hepatitis C virus 148Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg 1 5 1015 Glu Phe Asp Glu 20 149 20 PRT Hepatitis C virus 149 Val Asn Gln ArgAla Val Val Ala Pro Asp Lys Glu Val Leu Tyr Glu 1 5 10 15 Ala Phe AspGlu 20 150 20 PRT Hepatitis C virus 150 Ser Gln His Leu Pro Tyr Ile GluGln Gly Met Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys 20 151 20 PRTHepatitis C virus 151 Ala Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln ArgIle Ala Glu Met 1 5 10 15 Leu Lys Ser Lys 20 152 20 PRT Hepatitis Cvirus 152 Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln ThrAla 1 5 10 15 Ser Arg Gln Ala 20 153 20 PRT Hepatitis C virus 153 IleAla Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala 1 5 10 15Ser Lys Gln Ala 20 154 23 PRT Human immunodeficiency virus 154 Asn AsnThr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr 1 5 10 15 ThrThr Gly Glu Ile Ile Gly 20 155 23 PRT Human immunodeficiency virus 155Asn Asn Thr Arg Lys Ser Ile Tyr Ile Gly Pro Gly Arg Ala Phe His 1 5 1015 Thr Thr Gly Arg Ile Ile Gly 20 156 23 PRT Human immunodeficiencyvirus 156 Asn Asn Thr Thr Arg Ser Ile His Ile Gly Pro Gly Arg Ala PheTyr 1 5 10 15 Ala Thr Gly Asp Ile Ile Gly 20 157 23 PRT Humanimmunodeficiency virus 157 Tyr Asn Lys Arg Lys Arg Ile His Ile Gly ProGly Arg Ala Phe Tyr 1 5 10 15 Thr Thr Lys Asn Ile Ile Gly 20 158 23 PRTHuman immunodeficiency virus 158 Asn Asn Thr Arg Lys Ser Ile Thr Lys GlyPro Gly Arg Val Ile Tyr 1 5 10 15 Ala Thr Gly Gln Ile Ile Gly 20 159 22PRT Human immunodeficiency virus 159 Asn Asn Thr Arg Arg Gly Ile His PheGly Pro Gly Gln Ala Leu Tyr 1 5 10 15 Thr Thr Gly Ile Val Gly 20 160 24PRT Human immunodeficiency virus 160 Asn Asn Thr Arg Lys Ser Ile Arg IleGln Arg Gly Pro Gly Arg Ala 1 5 10 15 Phe Val Thr Ile Gly Lys Ile Gly 20161 22 PRT Human immunodeficiency virus 161 Gln Asn Thr Arg Gln Arg ThrPro Ile Gly Leu Gly Gln Ser Leu Tyr 1 5 10 15 Thr Thr Arg Ser Arg Ser 20162 21 PRT Human immunodeficiency virus 162 Gln Ile Asp Ile Gln Glu MetArg Ile Gly Pro Met Ala Trp Tyr Ser 1 5 10 15 Met Gly Ile Gly Gly 20 16323 PRT Human immunodeficiency virus 163 Asn Asn Thr Arg Arg Gly Ile HisMet Gly Trp Gly Arg Thr Phe Tyr 1 5 10 15 Ala Thr Gly Glu Ile Ile Gly 20164 22 PRT Human immunodeficiency virus 164 Asn Lys Thr Val Val Pro IleThr Leu Met Ser Gly Leu Val Phe His 1 5 10 15 Ser Gln Pro Ile Asn Lys 20165 22 PRT Human immunodeficiency virus 165 Asn Lys Thr Val Leu Pro ValThr Ile Met Ser Gly Leu Val Phe His 1 5 10 15 Ser Gln Pro Ile Asn Asp 20166 24 PRT Human T-cell lymphotropic virus 166 Gly Gly Val Leu Tyr SerPro Asn Val Ser Val Pro Ser Ser Ser Ser 1 5 10 15 Thr Leu Leu Tyr ProSer Leu Ala 20 167 23 PRT Human T-cell lymphotropic virus 167 Gly GlyTyr Thr Cys Ile Val Cys Ile Asp Arg Ala Ser Leu Ser Thr 1 5 10 15 TrpHis Val Leu Tyr Ser Pro 20 168 24 PRT Human T-cell lymphotropic virus168 Gly Gly Asn Ser Leu Ile Leu Pro Pro Phe Ser Leu Ser Pro Val Pro 1 510 15 Thr Leu Gly Ser Arg Ser Arg Arg 20 169 38 PRT Human T-celllymphotropic virus 169 Gly Gly Asp Ala Pro Gly Tyr Asp Pro Ile Trp PheLeu Asn Thr Glu 1 5 10 15 Pro Ser Gln Leu Pro Pro Thr Ala Pro Pro LeuLeu Pro His Ser Asn 20 25 30 Leu Asp His Ile Leu Glu 35 170 33 PRT HumanT-cell lymphotropic virus 170 Gly Gly Gln Tyr Ala Ala Gln Asn Arg ArgGly Leu Asp Leu Leu Phe 1 5 10 15 Trp Glu Gln Gly Gly Leu Cys Lys AlaLeu Gln Glu Gln Cys Arg Phe 20 25 30 Pro 171 33 PRT Human T-celllymphotropic virus 171 Gly Gly Pro Pro Pro Pro Ser Ser Pro Thr His AspPro Pro Asp Ser 1 5 10 15 Asp Pro Gln Ile Pro Pro Pro Tyr Val Glu ProThr Ala Pro Gln Val 20 25 30 Leu 172 20 PRT Human T-cell lymphotropicvirus 172 Gly Gly Lys Lys Pro Asn Arg Gln Gly Leu Gly Tyr Tyr Ser ProSer 1 5 10 15 Tyr Asn Asp Pro 20 173 38 PRT Human T-cell lymphotropicvirus 173 Gly Gly Asp Ala Pro Gly Tyr Asp Pro Leu Trp Phe Ile Thr SerGlu 1 5 10 15 Pro Thr Gln Pro Pro Pro Thr Ser Pro Pro Leu Val His AspSer Asp 20 25 30 Leu Glu His Val Leu Thr 35 174 40 PRT Human T-celllymphotropic virus 174 Gly Gly Tyr Ser Cys Met Val Cys Val Asp Arg SerSer Leu Ser Ser 1 5 10 15 Trp His Val Leu Tyr Thr Pro Asn Ile Ser IlePro Gln Gln Thr Ser 20 25 30 Ser Arg Thr Ile Leu Phe Pro Ser 35 40 17532 PRT Human T-cell lymphotropic virus 175 Gly Gly Pro Thr Thr Thr ProPro Pro Pro Pro Pro Pro Ser Pro Glu 1 5 10 15 Ala His Val Pro Pro ProTyr Val Glu Pro Thr Thr Thr Gln Cys Phe 20 25 30 176 13 PRT Humanimmunodeficiency virus 176 Gly Gly Gly Ile Trp Gly Cys Ser Gly Lys LeuIle Cys 1 5 10 177 12 PRT Human immunodeficiency virus 177 Ile Trp GlyCys Ser Gly Lys Leu Ile Cys Gly Gly 1 5 10 178 9 PRT Hepatitis C virus178 Met Ser Thr Ile Pro Lys Pro Gln Arg 1 5 179 9 PRT Hepatitis C virus179 Ser Thr Ile Pro Lys Pro Gln Arg Lys 1 5 180 9 PRT Hepatitis C virus180 Thr Ile Pro Lys Pro Gln Arg Lys Thr 1 5 181 9 PRT Hepatitis C virus181 Ile Pro Lys Pro Gln Arg Lys Thr Lys 1 5 182 9 PRT Hepatitis C virus182 Pro Lys Pro Gln Arg Lys Thr Lys Arg 1 5 183 9 PRT Hepatitis C virus183 Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 184 9 PRT Hepatitis C virus184 Pro Gln Arg Lys Thr Lys Arg Asn Thr 1 5 185 9 PRT Hepatitis C virus185 Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 186 9 PRT Hepatitis C virus186 Arg Lys Thr Lys Arg Asn Thr Asn Arg 1 5 187 9 PRT Hepatitis C virus187 Lys Thr Lys Arg Asn Thr Asn Arg Arg 1 5 188 9 PRT Hepatitis C virus188 Thr Lys Arg Asn Thr Asn Arg Arg Pro 1 5 189 9 PRT Hepatitis C virus189 Lys Arg Asn Thr Asn Arg Arg Pro Gln 1 5 190 9 PRT Hepatitis C virus190 Arg Asn Thr Asn Arg Arg Pro Gln Asp 1 5 191 9 PRT Hepatitis C virus191 Asn Thr Asn Arg Arg Pro Gln Asp Val 1 5 192 9 PRT Hepatitis C virus192 Thr Asn Arg Arg Pro Gln Asp Val Lys 1 5 193 9 PRT Hepatitis C virus193 Asn Arg Arg Pro Gln Asp Val Lys Phe 1 5 194 9 PRT Hepatitis C virus194 Arg Arg Pro Gln Asp Val Lys Phe Pro 1 5 195 9 PRT Hepatitis C virus195 Arg Pro Gln Asp Val Lys Phe Pro Gly 1 5 196 9 PRT Hepatitis C virus196 Pro Gln Asp Val Lys Phe Pro Gly Gly 1 5 197 9 PRT Hepatitis C virus197 Gln Asp Val Lys Phe Pro Gly Gly Gly 1 5 198 9 PRT Hepatitis C virus198 Asp Val Lys Phe Pro Gly Gly Gly Gln 1 5 199 9 PRT Hepatitis C virus199 Val Lys Phe Pro Gly Gly Gly Gln Ile 1 5 200 9 PRT Hepatitis C virus200 Lys Phe Pro Gly Gly Gly Gln Ile Val 1 5 201 9 PRT Hepatitis C virus201 Phe Pro Gly Gly Gly Gln Ile Val Gly 1 5 202 9 PRT Hepatitis C virus202 Pro Gly Gly Gly Gln Ile Val Gly Gly 1 5 203 9 PRT Hepatitis C virus203 Gly Gly Gly Gln Ile Val Gly Gly Val 1 5 204 9 PRT Hepatitis C virus204 Gly Gly Gln Ile Val Gly Gly Val Tyr 1 5 205 9 PRT Hepatitis C virus205 Gly Gln Ile Val Gly Gly Val Tyr Leu 1 5 206 9 PRT Hepatitis C virus206 Gln Ile Val Gly Gly Val Tyr Leu Leu 1 5 207 9 PRT Hepatitis C virus207 Ile Val Gly Gly Val Tyr Leu Leu Pro 1 5 208 9 PRT Hepatitis C virus208 Val Gly Gly Val Tyr Leu Leu Pro Arg 1 5 209 9 PRT Hepatitis C virus209 Gly Gly Val Tyr Leu Leu Pro Arg Arg 1 5 210 9 PRT Hepatitis C virus210 Gly Val Tyr Leu Leu Pro Arg Arg Gly 1 5 211 9 PRT Hepatitis C virus211 Val Tyr Leu Leu Pro Arg Arg Gly Pro 1 5 212 9 PRT Hepatitis C virus212 Tyr Leu Leu Pro Arg Arg Gly Pro Arg 1 5 213 9 PRT Hepatitis C virus213 Leu Leu Pro Arg Arg Gly Pro Arg Leu 1 5 214 9 PRT Hepatitis C virus214 Leu Pro Arg Arg Gly Pro Arg Leu Gly 1 5 215 9 PRT Hepatitis C virus215 Pro Arg Arg Gly Pro Arg Leu Gly Val 1 5 216 9 PRT Hepatitis C virus216 Arg Arg Gly Pro Arg Leu Gly Val Arg 1 5 217 9 PRT Hepatitis C virus217 Arg Gly Pro Arg Leu Gly Val Arg Ala 1 5 218 9 PRT Hepatitis C virus218 Gly Pro Arg Leu Gly Val Arg Ala Thr 1 5 219 9 PRT Hepatitis C virus219 Pro Arg Leu Gly Val Arg Ala Thr Arg 1 5 220 9 PRT Hepatitis C virus220 Arg Leu Gly Val Arg Ala Thr Arg Lys 1 5 221 9 PRT Hepatitis C virus221 Leu Gly Val Arg Ala Thr Arg Lys Thr 1 5 222 9 PRT Hepatitis C virus222 Gly Val Arg Ala Thr Arg Lys Thr Ser 1 5 223 9 PRT Hepatitis C virus223 Val Arg Ala Thr Arg Lys Thr Ser Glu 1 5 224 9 PRT Hepatitis C virus224 Arg Ala Thr Arg Lys Thr Ser Glu Arg 1 5 225 9 PRT Hepatitis C virus225 Ala Thr Arg Lys Thr Ser Glu Arg Ser 1 5 226 9 PRT Hepatitis C virus226 Thr Arg Lys Thr Ser Glu Arg Ser Gln 1 5 227 9 PRT Hepatitis C virus227 Arg Lys Thr Ser Glu Arg Ser Gln Pro 1 5 228 9 PRT Hepatitis C virus228 Lys Thr Ser Glu Arg Ser Gln Pro Arg 1 5 229 9 PRT Hepatitis C virus229 Thr Ser Glu Arg Ser Gln Pro Arg Gly 1 5 230 9 PRT Hepatitis C virus230 Ser Glu Arg Ser Gln Pro Arg Gly Arg 1 5 231 9 PRT Hepatitis C virus231 Glu Arg Ser Gln Pro Arg Gly Arg Arg 1 5 232 9 PRT Hepatitis C virus232 Arg Ser Gln Pro Arg Gly Arg Arg Gln 1 5 233 9 PRT Hepatitis C virus233 Ser Gln Pro Arg Gly Arg Arg Gln Pro 1 5 234 9 PRT Hepatitis C virus234 Gln Pro Arg Gly Arg Arg Gln Pro Ile 1 5 235 9 PRT Hepatitis C virus235 Pro Arg Gly Arg Arg Gln Pro Ile Pro 1 5 236 9 PRT Hepatitis C virus236 Arg Gly Arg Arg Gln Pro Ile Pro Lys 1 5 237 9 PRT Hepatitis C virus237 Gly Arg Arg Gln Pro Ile Pro Lys Val 1 5 238 9 PRT Hepatitis C virus238 Arg Arg Gln Pro Ile Pro Lys Val Arg 1 5 239 9 PRT Hepatitis C virus239 Arg Gln Pro Ile Pro Lys Val Arg Arg 1 5 240 9 PRT Hepatitis C virus240 Gln Pro Ile Pro Lys Val Arg Arg Pro 1 5 241 9 PRT Hepatitis C virus241 Pro Ile Pro Lys Val Arg Arg Pro Glu 1 5 242 9 PRT Hepatitis C virus242 Ile Pro Lys Val Arg Arg Pro Glu Gly 1 5 243 9 PRT Hepatitis C virus243 Pro Lys Val Arg Arg Pro Glu Gly Arg 1 5 244 9 PRT Hepatitis C virus244 Lys Val Arg Arg Pro Glu Gly Arg Thr 1 5 245 9 PRT Hepatitis C virus245 Val Arg Arg Pro Glu Gly Arg Thr Trp 1 5 246 9 PRT Hepatitis C virus246 Arg Arg Pro Glu Gly Arg Thr Trp Ala 1 5 247 9 PRT Hepatitis C virus247 Arg Pro Glu Gly Arg Thr Trp Ala Gln 1 5 248 9 PRT Hepatitis C virus248 Pro Glu Gly Arg Thr Trp Ala Gln Pro 1 5 249 9 PRT Hepatitis C virus249 Glu Gly Arg Thr Trp Ala Gln Pro Gly 1 5 250 9 PRT Hepatitis C virus250 Gly Arg Thr Trp Ala Gln Pro Gly Tyr 1 5 251 9 PRT Hepatitis C virus251 Arg Thr Trp Ala Gln Pro Gly Tyr Pro 1 5 252 9 PRT Hepatitis C virus252 Thr Trp Ala Gln Pro Gly Tyr Pro Trp 1 5 253 9 PRT Hepatitis C virus253 Trp Ala Gln Pro Gly Tyr Pro Trp Pro 1 5 254 9 PRT Hepatitis C virus254 Ala Gln Pro Gly Tyr Pro Trp Pro Leu 1 5 255 9 PRT Hepatitis C virus255 Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 1 5 256 9 PRT Hepatitis C virus256 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly 1 5 257 9 PRT Hepatitis C virus257 Gly Tyr Pro Trp Pro Leu Tyr Gly Asn 1 5 258 9 PRT Hepatitis C virus258 Leu Ser Gly Lys Pro Ala Ile Ile Pro 1 5 259 9 PRT Hepatitis C virus259 Ser Gly Lys Pro Ala Ile Ile Pro Asp 1 5 260 9 PRT Hepatitis C virus260 Gly Lys Pro Ala Ile Ile Pro Asp Arg 1 5 261 9 PRT Hepatitis C virus261 Lys Pro Ala Ile Ile Pro Asp Arg Glu 1 5 262 9 PRT Hepatitis C virus262 Pro Ala Ile Ile Pro Asp Arg Glu Val 1 5 263 9 PRT Hepatitis C virus263 Ala Ile Ile Pro Asp Arg Glu Val Leu 1 5 264 9 PRT Hepatitis C virus264 Ile Ile Pro Asp Arg Glu Val Leu Tyr 1 5 265 9 PRT Hepatitis C virus265 Ile Pro Asp Arg Glu Val Leu Tyr Arg 1 5 266 9 PRT Hepatitis C virus266 Pro Asp Arg Glu Val Leu Tyr Arg Glu 1 5 267 9 PRT Hepatitis C virus267 Asp Arg Glu Val Leu Tyr Arg Glu Phe 1 5 268 9 PRT Hepatitis C virus268 Arg Glu Val Leu Tyr Arg Glu Phe Asp 1 5 269 9 PRT Hepatitis C virus269 Glu Val Leu Tyr Arg Glu Phe Asp Glu 1 5 270 9 PRT Hepatitis C virus270 Val Leu Tyr Arg Glu Phe Asp Glu Met 1 5 271 9 PRT Hepatitis C virus271 Leu Tyr Arg Glu Phe Asp Glu Met Glu 1 5 272 9 PRT Hepatitis C virus272 Tyr Arg Glu Phe Asp Glu Met Glu Glu 1 5 273 9 PRT Hepatitis C virus273 Arg Glu Phe Asp Glu Met Glu Glu Cys 1 5 274 9 PRT Hepatitis C virus274 Glu Phe Asp Glu Met Glu Glu Cys Ser 1 5 275 9 PRT Hepatitis C virus275 Phe Asp Glu Met Glu Glu Cys Ser Gln 1 5 276 9 PRT Hepatitis C virus276 Asp Glu Met Glu Glu Cys Ser Gln His 1 5 277 9 PRT Hepatitis C virus277 Glu Met Glu Glu Cys Ser Gln His Leu 1 5 278 9 PRT Hepatitis C virus278 Met Glu Glu Cys Ser Gln His Leu Pro 1 5 279 9 PRT Hepatitis C virus279 Glu Glu Cys Ser Gln His Leu Pro Tyr 1 5 280 9 PRT Hepatitis C virus280 Glu Cys Ser Gln His Leu Pro Tyr Ile 1 5 281 9 PRT Hepatitis C virus281 Cys Ser Gln His Leu Pro Tyr Ile Glu 1 5 282 9 PRT Hepatitis C virus282 Ser Gln His Leu Pro Tyr Ile Glu Gln 1 5 283 9 PRT Hepatitis C virus283 Gln His Leu Pro Tyr Ile Glu Gln Gly 1 5 284 9 PRT Hepatitis C virus284 His Leu Pro Tyr Ile Glu Gln Gly Met 1 5 285 9 PRT Hepatitis C virus285 Leu Pro Tyr Ile Glu Gln Gly Met Met 1 5 286 9 PRT Hepatitis C virus286 Pro Tyr Ile Glu Gln Gly Met Met Leu 1 5 287 9 PRT Hepatitis C virus287 Tyr Ile Glu Gln Gly Met Met Leu Ala 1 5 288 9 PRT Hepatitis C virus288 Ile Glu Gln Gly Met Met Leu Ala Glu 1 5 289 9 PRT Hepatitis C virus289 Glu Gln Gly Met Met Leu Ala Glu Gln 1 5 290 9 PRT Hepatitis C virus290 Gln Gly Met Met Leu Ala Glu Gln Phe 1 5 291 9 PRT Hepatitis C virus291 Gly Met Met Leu Ala Glu Gln Phe Lys 1 5 292 9 PRT Hepatitis C virus292 Met Met Leu Ala Glu Gln Phe Lys Gln 1 5 293 9 PRT Hepatitis C virus293 Met Leu Ala Glu Gln Phe Lys Gln Lys 1 5 294 9 PRT Hepatitis C virus294 Leu Ala Glu Gln Phe Lys Gln Lys Ala 1 5 295 9 PRT Hepatitis C virus295 Ala Glu Gln Phe Lys Gln Lys Ala Leu 1 5 296 9 PRT Hepatitis C virus296 Glu Gln Phe Lys Gln Lys Ala Leu Gly 1 5 297 9 PRT Hepatitis C virus297 Gln Phe Lys Gln Lys Ala Leu Gly Leu 1 5 298 9 PRT Hepatitis C virus298 Phe Lys Gln Lys Ala Leu Gly Leu Leu 1 5 299 9 PRT Hepatitis C virus299 Lys Gln Lys Ala Leu Gly Leu Leu Gln 1 5 300 9 PRT Hepatitis C virus300 Gln Lys Ala Leu Gly Leu Leu Gln Thr 1 5 301 9 PRT Hepatitis C virus301 Lys Ala Leu Gly Leu Leu Gln Thr Ala 1 5 302 9 PRT Hepatitis C virus302 Ala Leu Gly Leu Leu Gln Thr Ala Ser 1 5 303 9 PRT Hepatitis C virus303 Leu Gly Leu Leu Gln Thr Ala Ser Arg 1 5 304 9 PRT Hepatitis C virus304 Gly Leu Leu Gln Thr Ala Ser Arg Gln 1 5 305 9 PRT Hepatitis C virus305 Leu Leu Gln Thr Ala Ser Arg Gln Ala 1 5 306 9 PRT Hepatitis C virus306 Leu Gln Thr Ala Ser Arg Gln Ala Glu 1 5 307 9 PRT Hepatitis C virus307 Gln Thr Ala Ser Arg Gln Ala Glu Val 1 5 308 9 PRT Hepatitis C virus308 Thr Ala Ser Arg Gln Ala Glu Val Ile 1 5 309 9 PRT Hepatitis C virus309 Ala Ser Arg Gln Ala Glu Val Ile Ala 1 5 310 9 PRT Hepatitis C virus310 Ser Arg Gln Ala Glu Val Ile Ala Pro 1 5 311 9 PRT Hepatitis C virus311 Arg Gln Ala Glu Val Ile Ala Pro Ala 1 5 312 9 PRT Hepatitis C virus312 Gln Ala Glu Val Ile Ala Pro Ala Val 1 5 313 9 PRT Hepatitis C virus313 Ala Glu Val Ile Ala Pro Ala Val Gln 1 5 314 9 PRT Hepatitis C virus314 Glu Val Ile Ala Pro Ala Val Gln Thr 1 5 315 9 PRT Hepatitis C virus315 Val Ile Ala Pro Ala Val Gln Thr Asn 1 5 316 9 PRT Hepatitis C virus316 Ile Ala Pro Ala Val Gln Thr Asn Trp 1 5 317 9 PRT Hepatitis C virus317 Ala Pro Ala Val Gln Thr Asn Trp Gln 1 5 318 9 PRT Hepatitis C virus318 Gly Asn Ile Thr Arg Tyr Glu Ser Glu 1 5 319 9 PRT Hepatitis C virus319 Asn Ile Thr Arg Tyr Glu Ser Glu Asn 1 5 320 9 PRT Hepatitis C virus320 Ile Thr Arg Tyr Glu Ser Glu Asn Lys 1 5 321 9 PRT Hepatitis C virus321 Thr Arg Tyr Glu Ser Glu Asn Lys Val 1 5 322 9 PRT Hepatitis C virus322 Arg Tyr Glu Ser Glu Asn Lys Val Val 1 5 323 9 PRT Hepatitis C virus323 Tyr Glu Ser Glu Asn Lys Val Val Ile 1 5 324 9 PRT Hepatitis C virus324 Glu Ser Glu Asn Lys Val Val Ile Leu 1 5 325 9 PRT Hepatitis C virus325 Ser Glu Asn Lys Val Val Ile Leu Asp 1 5 326 9 PRT Hepatitis C virus326 Glu Asn Lys Val Val Ile Leu Asp Ser 1 5 327 9 PRT Hepatitis C virus327 Asn Lys Val Val Ile Leu Asp Ser Phe 1 5 328 9 PRT Hepatitis C virus328 Lys Val Val Ile Leu Asp Ser Phe Asp 1 5 329 9 PRT Hepatitis C virus329 Val Val Ile Leu Asp Ser Phe Asp Pro 1 5 330 9 PRT Hepatitis C virus330 Val Ile Leu Asp Ser Phe Asp Pro Leu 1 5 331 9 PRT Hepatitis C virus331 Ile Leu Asp Ser Phe Asp Pro Leu Val 1 5 332 9 PRT Hepatitis C virus332 Leu Asp Ser Phe Asp Pro Leu Val Ala 1 5 333 9 PRT Hepatitis C virus333 Asp Ser Phe Asp Pro Leu Val Ala Glu 1 5 334 9 PRT Hepatitis C virus334 Ser Phe Asp Pro Leu Val Ala Glu Glu 1 5 335 9 PRT Hepatitis C virus335 Phe Asp Pro Leu Val Ala Glu Glu Asp 1 5 336 9 PRT Hepatitis C virus336 Asp Pro Leu Val Ala Glu Glu Asp Glu 1 5 337 9 PRT Hepatitis C virus337 Pro Leu Val Ala Glu Glu Asp Glu Arg 1 5 338 9 PRT Hepatitis C virus338 Leu Val Ala Glu Glu Asp Glu Arg Glu 1 5 339 9 PRT Hepatitis C virus339 Val Ala Glu Glu Asp Glu Arg Glu Ile 1 5 340 9 PRT Hepatitis C virus340 Ala Glu Glu Asp Glu Arg Glu Ile Ser 1 5 341 9 PRT Hepatitis C virus341 Glu Glu Asp Glu Arg Glu Ile Ser Val 1 5 342 9 PRT Hepatitis C virus342 Glu Asp Glu Arg Glu Ile Ser Val Pro 1 5 343 9 PRT Hepatitis C virus343 Asp Glu Arg Glu Ile Ser Val Pro Ala 1 5 344 9 PRT Hepatitis C virus344 Glu Arg Glu Ile Ser Val Pro Ala Glu 1 5 345 9 PRT Hepatitis C virus345 Arg Glu Ile Ser Val Pro Ala Glu Ile 1 5 346 9 PRT Hepatitis C virus346 Glu Ile Ser Val Pro Ala Glu Ile Leu 1 5 347 9 PRT Hepatitis C virus347 Ile Ser Val Pro Ala Glu Ile Leu Arg 1 5 348 9 PRT Hepatitis C virus348 Ser Val Pro Ala Glu Ile Leu Arg Lys 1 5 349 9 PRT Hepatitis C virus349 Val Pro Ala Glu Ile Leu Arg Lys Ser 1 5 350 9 PRT Hepatitis C virus350 Pro Ala Glu Ile Leu Arg Lys Ser Arg 1 5 351 9 PRT Hepatitis C virus351 Ala Glu Ile Leu Arg Lys Ser Arg Arg 1 5 352 9 PRT Hepatitis C virus352 Glu Ile Leu Arg Lys Ser Arg Arg Phe 1 5 353 9 PRT Hepatitis C virus353 Ile Leu Arg Lys Ser Arg Arg Phe Ala 1 5 354 9 PRT Hepatitis C virus354 Leu Arg Lys Ser Arg Arg Phe Ala Gln 1 5 355 9 PRT Hepatitis C virus355 Arg Lys Ser Arg Arg Phe Ala Gln Ala 1 5 356 9 PRT Hepatitis C virus356 Lys Ser Arg Arg Phe Ala Gln Ala Leu 1 5 357 9 PRT Hepatitis C virus357 Ser Arg Arg Phe Ala Gln Ala Leu Pro 1 5 358 9 PRT Hepatitis C virus358 Arg Arg Phe Ala Gln Ala Leu Pro Val 1 5 359 9 PRT Hepatitis C virus359 Arg Phe Ala Gln Ala Leu Pro Val Trp 1 5 360 9 PRT Hepatitis C virus360 Phe Ala Gln Ala Leu Pro Val Trp Ala 1 5 361 9 PRT Hepatitis C virus361 Ala Gln Ala Leu Pro Val Trp Ala Arg 1 5 362 9 PRT Hepatitis C virus362 Gln Ala Leu Pro Val Trp Ala Arg Pro 1 5 363 9 PRT Hepatitis C virus363 Ala Leu Pro Val Trp Ala Arg Pro Asp 1 5 364 9 PRT Hepatitis C virus364 Leu Pro Val Trp Ala Arg Pro Asp Tyr 1 5 365 9 PRT Hepatitis C virus365 Pro Val Trp Ala Arg Pro Asp Tyr Asn 1 5 366 9 PRT Hepatitis C virus366 Val Trp Ala Arg Pro Asp Tyr Asn Pro 1 5 367 9 PRT Hepatitis C virus367 Trp Ala Arg Pro Asp Tyr Asn Pro Pro 1 5 368 9 PRT Hepatitis C virus368 Ala Arg Pro Asp Tyr Asn Pro Pro Leu 1 5 369 9 PRT Hepatitis C virus369 Arg Pro Asp Tyr Asn Pro Pro Leu Val 1 5 370 9 PRT Hepatitis C virus370 Pro Asp Tyr Asn Pro Pro Leu Val Glu 1 5 371 9 PRT Hepatitis C virus371 Asp Tyr Asn Pro Pro Leu Val Glu Thr 1 5 372 9 PRT Hepatitis C virus372 Tyr Asn Pro Pro Leu Val Glu Thr Trp 1 5 373 9 PRT Hepatitis C virus373 Asn Pro Pro Leu Val Glu Thr Trp Lys 1 5 374 9 PRT Hepatitis C virus374 Pro Pro Leu Val Glu Thr Trp Lys Lys 1 5 375 9 PRT Hepatitis C virus375 Pro Leu Val Glu Thr Trp Lys Lys Pro 1 5 376 9 PRT Hepatitis C virus376 Leu Val Glu Thr Trp Lys Lys Pro Asp 1 5 377 9 PRT Hepatitis C virus377 Val Glu Thr Trp Lys Lys Pro Asp Tyr 1 5 378 9 PRT Hepatitis C virus378 Glu Thr Trp Lys Lys Pro Asp Tyr Glu 1 5 379 9 PRT Hepatitis C virus379 Thr Trp Lys Lys Pro Asp Tyr Glu Pro 1 5 380 9 PRT Hepatitis C virus380 Trp Lys Lys Pro Asp Tyr Glu Pro Pro 1 5 381 9 PRT Hepatitis C virus381 Lys Lys Pro Asp Tyr Glu Pro Pro Val 1 5 382 9 PRT Hepatitis C virus382 Lys Pro Asp Tyr Glu Pro Pro Val Val 1 5 383 9 PRT Hepatitis C virus383 Lys Pro Asp Tyr Glu Pro Pro Val Val 1 5 384 9 PRT Hepatitis C virus384 Asp Tyr Glu Pro Pro Val Val His Gly 1 5 385 9 PRT Hepatitis C virus385 Tyr Glu Pro Pro Val Val His Gly Cys 1 5 386 9 PRT Hepatitis C virus386 Glu Pro Pro Val Val His Gly Cys Pro 1 5 387 9 PRT Hepatitis C virus387 Pro Pro Val Val His Gly Cys Pro Leu 1 5 388 9 PRT Hepatitis C virus388 Pro Val Val His Gly Cys Pro Leu Pro 1 5 389 9 PRT Hepatitis C virus389 Val Val His Gly Cys Pro Leu Pro Pro 1 5 390 9 PRT Hepatitis C virus390 Val His Gly Cys Pro Leu Pro Pro Pro 1 5 391 9 PRT Hepatitis C virus391 His Gly Cys Pro Leu Pro Pro Pro Lys 1 5 392 9 PRT Hepatitis C virus392 Gly Cys Pro Leu Pro Pro Pro Lys Ser 1 5 393 9 PRT Hepatitis C virus393 Cys Pro Leu Pro Pro Pro Lys Ser Pro 1 5 394 9 PRT Hepatitis C virus394 Pro Leu Pro Pro Pro Lys Ser Pro Pro 1 5 395 9 PRT Hepatitis C virus395 Leu Pro Pro Pro Lys Ser Pro Pro Val 1 5 396 9 PRT Hepatitis C virus396 Pro Pro Pro Lys Ser Pro Pro Val Pro 1 5 397 9 PRT Hepatitis C virus397 Pro Pro Lys Ser Pro Pro Val Pro Pro 1 5 398 9 PRT Hepatitis C virus398 Pro Lys Ser Pro Pro Val Pro Pro Pro 1 5 399 9 PRT Hepatitis C virus399 Lys Ser Pro Pro Val Pro Pro Pro Arg 1 5 400 9 PRT Hepatitis C virus400 Ser Pro Pro Val Pro Pro Pro Arg Lys 1 5 401 9 PRT Hepatitis C virus401 Pro Pro Val Pro Pro Pro Arg Lys Lys 1 5 402 32 PRT Hepatitis C virus402 Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 510 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 2025 30 403 39 PRT Hepatitis C virus 403 Gly Gly Val Tyr Leu Leu Pro ArgArg Gly Pro Arg Leu Gly Val Arg 1 5 10 15 Ala Thr Arg Lys Thr Ser GluArg Ser Gln Pro Arg Gly Arg Arg Gln 20 25 30 Pro Ile Pro Glu Val Arg Arg35 404 32 PRT Hepatitis C virus 404 Ser Gln His Leu Pro Tyr Ile Glu GlnGly Met Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys Ala Leu Gly LeuLeu Gln Thr Ala Ser Arg Gln Ala 20 25 30 405 32 PRT Hepatitis C virus405 Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser 1 510 15 Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn 2025 30 406 22 PRT Hepatitis C virus 406 Gly Glu Thr Tyr Thr Ser Gly GlyAla Ala Ser His Thr Thr Ser Thr 1 5 10 15 Leu Ala Ser Leu Phe Ser 20 40724 PRT Hepatitis C virus 407 Ser His Thr Thr Ser Thr Leu Ala Ser Leu PheSer Pro Gly Ala Ser 1 5 10 15 Gln Arg Ile Gln Leu Val Asn Thr 20 408 22PRT Hepatitis C virus 408 Gly His Thr Arg Val Ser Gly Gly Ala Ala AlaSer Asp Thr Arg Gly 1 5 10 15 Leu Val Ser Leu Phe Ser 20 409 24 PRTHepatitis C virus 409 Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe SerPro Gly Ser Ala 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 410 22 PRTHepatitis C virus 410 Gly His Thr Arg Val Thr Gly Gly Val Gln Gly HisVal Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu Phe Arg 20 411 24 PRTHepatitis C virus 411 Gly His Val Thr Cys Thr Leu Thr Ser Leu Phe ArgPro Gly Ala Ser 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 412 22 PRTHepatitis C virus 412 Gly His Thr His Val Thr Gly Gly Arg Val Ala SerSer Thr Gln Ser 1 5 10 15 Leu Val Ser Trp Leu Ser 20 413 24 PRTHepatitis C virus 413 Ala Ser Ser Thr Gln Ser Leu Val Ser Trp Leu SerGln Gly Pro Ser 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 414 22 PRTHepatitis C virus 414 Gly Asp Thr His Val Thr Gly Gly Ala Gln Ala LysThr Thr Asn Arg 1 5 10 15 Leu Val Ser Met Phe Ala 20 415 24 PRTHepatitis C virus 415 Ala Lys Thr Thr Asn Arg Leu Val Ser Met Phe AlaSer Gly Pro Ser 1 5 10 15 Gln Lys Ile Gln Leu Ile Asn Thr 20 416 22 PRTHepatitis C virus 416 Ala Glu Thr Tyr Thr Ser Gly Gly Asn Ala Gly HisThr Met Thr Gly 1 5 10 15 Ile Val Arg Phe Phe Ala 20 417 24 PRTHepatitis C virus 417 Gly His Thr Met Thr Gly Ile Val Arg Phe Phe AlaPro Gly Pro Lys 1 5 10 15 Gln Asn Val His Leu Ile Asn Thr 20 418 22 PRTHepatitis C virus 418 Ala Glu Thr Ile Val Ser Gly Gly Gln Ala Ala ArgAla Met Ser Gly 1 5 10 15 Leu Val Ser Leu Phe Thr 20 419 24 PRTHepatitis C virus 419 Ala Arg Ala Met Ser Gly Leu Val Ser Leu Phe ThrPro Gly Ala Lys 1 5 10 15 Gln Asn Ile Gln Leu Ile Asn Thr 20 420 22 PRTHepatitis C virus 420 Ala Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala ArgThr Thr Gln Gly 1 5 10 15 Leu Val Ser Leu Phe Ser 20 421 24 PRTHepatitis C virus 421 Ala Arg Thr Thr Gln Gly Leu Val Ser Leu Phe SerArg Gly Ala Lys 1 5 10 15 Gln Asp Ile Gln Leu Ile Asn Thr 20 422 6 PRTHepatitis C virus 422 Pro Gln Arg Lys Thr Lys 1 5 423 7 PRT Hepatitis Cvirus 423 Lys Thr Lys Arg Asn Thr Asn 1 5 424 7 PRT Hepatitis C virus424 Pro Gln Asp Val Lys Phe Pro 1 5 425 6 PRT Hepatitis C virus 425 TyrLeu Leu Pro Arg Arg 1 5 426 7 PRT Hepatitis C virus 426 Pro Arg Arg GlyPro Arg Leu 1 5 427 7 PRT Hepatitis C virus 427 Arg Leu Gly Val Arg AlaThr 1 5 428 7 PRT Hepatitis C virus 428 Ser Gln Pro Arg Gly Arg Arg 1 5429 7 PRT Hepatitis C virus 429 Arg Arg Gln Pro Ile Pro Lys 1 5 430 6PRT Hepatitis C virus 430 Arg Thr Trp Ala Gln Pro 1 5 431 8 PRTHepatitis C virus 431 Gln Pro Gly Tyr Pro Trp Pro Leu 1 5 432 6 PRTHepatitis C virus 432 Pro Asp Arg Glu Val Leu 1 5 433 6 PRT Hepatitis Cvirus 433 His Leu Pro Tyr Ile Glu 1 5 434 8 PRT Hepatitis C virus 434Tyr Ile Glu Gln Gly Met Met Leu 1 5 435 7 PRT Hepatitis C virus 435 AlaGlu Gln Phe Lys Gln Lys 1 5 436 6 PRT Hepatitis C virus 436 Lys Gln LysAla Leu Gly 1 5 437 7 PRT Hepatitis C virus 437 Leu Gly Leu Leu Gln ThrAla 1 5 438 7 PRT Hepatitis C virus 438 Pro Ala Glu Ile Leu Arg Lys 1 5439 7 PRT Hepatitis C virus 439 Glu Ile Leu Arg Lys Ser Arg 1 5 440 7PRT Hepatitis C virus 440 Gln Ala Leu Pro Val Trp Ala 1 5 441 6 PRTHepatitis C virus 441 Pro Asp Tyr Asn Pro Pro 1 5 442 7 PRT Hepatitis Cvirus 442 Leu Val Glu Thr Trp Lys Lys 1 5 443 6 PRT Hepatitis C virus443 Asp Tyr Glu Pro Pro Val 1 5 444 5 PRT Hepatitis C virus 444 His GlyCys Pro Leu 1 5 445 20 PRT Hepatitis C virus 445 Gly Ala Leu Val Ala PheLys Ile Met Ser Gly Glu Val Pro Ser Thr 1 5 10 15 Glu Asp Leu Val 20 44620 PRT Hepatitis C virus 446 Val Pro Ser Thr Glu Asp Leu Val Asn Leu LeuPro Ala Ile Leu Ser 1 5 10 15 Pro Gly Ala Leu 20 447 20 PRT Hepatitis Cvirus 447 Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys AlaAla 1 5 10 15 Ile Leu Arg Arg 20 448 20 PRT Hepatitis C virus 448 ValCys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala 1 5 10 15Val Gln Trp Met 20 449 20 PRT Hepatitis C virus 449 Gly Glu Gly Ala ValGln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser 1 5 10 15 Arg Gly Asn His 20450 34 PRT Hepatitis C virus 450 Gly Gly Ile Pro Asp Arg Glu Val Leu TyrArg Gly Gly Lys Lys Pro 1 5 10 15 Asp Thr Tyr Glu Pro Pro Val Gly GlyArg Arg Pro Gln Asp Val Lys 20 25 30 Phe Pro 451 33 PRT Hepatitis Cvirus 451 Gly Gly Trp Ala Arg Pro Asp Tyr Asn Pro Pro Gly Gly Gln PheLys 1 5 10 15 Gln Lys Ala Leu Gly Leu Gly Ser Gly Val Tyr Leu Leu ProArg Arg 20 25 30 Gly 452 33 PRT Hepatitis C virus 452 Gly Gly Arg GlyArg Arg Gln Pro Ile Pro Lys Gly Gly Ser Gln His 1 5 10 15 Leu Pro TyrIle Glu Gln Ser Gly Pro Val Val His Gly Cys Pro Leu 20 25 30 Pro 453 34PRT Hepatitis C virus 453 Gly Glu Thr Tyr Thr Ser Gly Gly Ala Ala SerHis Thr Thr Ser Thr 1 5 10 15 Leu Ala Ser Leu Phe Ser Pro Gly Ala SerGln Arg Ile Gln Leu Val 20 25 30 Asn Thr 454 34 PRT Hepatitis C virus454 Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly 1 510 15 Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val 2025 30 Asn Thr 455 34 PRT Hepatitis C virus 455 Gly His Thr Arg Val ThrGly Gly Val Gln Gly His Val Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu PheArg Pro Gly Ala Ser Gln Lys Ile Gln Leu Val 20 25 30 Asn Thr 456 34 PRTHepatitis C virus 456 Gly His Thr His Val Thr Gly Gly Arg Val Ala SerSer Thr Gln Ser 1 5 10 15 Leu Val Ser Trp Leu Ser Gln Gly Pro Ser GlnLys Ile Gln Leu Val 20 25 30 Asn Thr 457 34 PRT Hepatitis C virus 457Gly Asp Thr His Val Thr Gly Gly Ala Gln Ala Lys Thr Thr Asn Arg 1 5 1015 Leu Val Ser Met Phe Ala Ser Gly Pro Ser Gln Lys Ile Gln Leu Ile 20 2530 Asn Thr 458 34 PRT Hepatitis C virus 458 Ala Glu Thr Tyr Thr Ser GlyGly Asn Ala Gly His Thr Met Thr Gly 1 5 10 15 Ile Val Arg Phe Phe AlaPro Gly Pro Lys Gln Asn Val His Leu Ile 20 25 30 Asn Thr 459 34 PRTHepatitis C virus 459 Ala Glu Thr Ile Val Ser Gly Gly Gln Ala Ala ArgAla Met Ser Gly 1 5 10 15 Leu Val Ser Leu Phe Thr Pro Gly Ala Lys GlnAsn Ile Gln Leu Ile 20 25 30 Asn Thr 460 34 PRT Hepatitis C virus 460Ala Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gln Gly 1 5 1015 Leu Val Ser Leu Phe Ser Arg Gly Ala Lys Gln Asp Ile Gln Leu Ile 20 2530 Asn Thr 461 22 PRT Hepatitis C virus 461 Gly Glu Thr Tyr Thr Ser GlyGly Ala Ala Ser His Thr Thr Ser Thr 1 5 10 15 Leu Ala Ser Leu Phe Ser 20462 24 PRT Hepatitis C virus 462 Ser His Thr Thr Ser Thr Leu Ala Ser LeuPhe Ser Pro Gly Ala Ser 1 5 10 15 Gln Arg Ile Gln Leu Val Asn Thr 20 46322 PRT Hepatitis C virus 463 Gly His Thr Arg Val Ser Gly Gly Ala Ala AlaSer Asp Thr Arg Gly 1 5 10 15 Leu Val Ser Leu Phe Ser 20 464 24 PRTHepatitis C virus 464 Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe SerPro Gly Ser Ala 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 465 22 PRTHepatitis C virus 465 Gly His Thr Arg Val Thr Gly Gly Val Gln Gly HisVal Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu Phe Arg 20 466 24 PRTHepatitis C virus 466 Gly His Val Thr Cys Thr Leu Thr Ser Leu Phe ArgPro Gly Ala Ser 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 467 22 PRTHepatitis C virus 467 Gly His Thr His Val Thr Gly Gly Arg Val Ala SerSer Thr Gln Ser 1 5 10 15 Leu Val Ser Trp Leu Ser 20 468 24 PRTHepatitis C virus 468 Ala Ser Ser Thr Gln Ser Leu Val Ser Trp Leu SerGln Gly Pro Ser 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 469 22 PRTHepatitis C virus 469 Gly Asp Thr His Val Thr Gly Gly Ala Gln Ala LysThr Thr Asn Arg 1 5 10 15 Leu Val Ser Met Phe Ala 20 470 24 PRTHepatitis C virus 470 Ala Lys Thr Thr Asn Arg Leu Val Ser Met Phe AlaSer Gly Pro Ser 1 5 10 15 Gln Lys Ile Gln Leu Ile Asn Thr 20 471 22 PRTHepatitis C virus 471 Ala Glu Thr Tyr Thr Ser Gly Gly Asn Ala Gly HisThr Met Thr Gly 1 5 10 15 Ile Val Arg Phe Phe Ala 20 472 24 PRTHepatitis C virus 472 Gly His Thr Met Thr Gly Ile Val Arg Phe Phe AlaPro Gly Pro Lys 1 5 10 15 Gln Asn Val His Leu Ile Asn Thr 20 473 22 PRTHepatitis C virus 473 Ala Glu Thr Ile Val Ser Gly Gly Gln Ala Ala ArgAla Met Ser Gly 1 5 10 15 Leu Val Ser Leu Phe Thr 20 474 24 PRTHepatitis C virus 474 Ala Arg Ala Met Ser Gly Leu Val Ser Leu Phe ThrPro Gly Ala Lys 1 5 10 15 Gln Asn Ile Gln Leu Ile Asn Thr 20 475 22 PRTHepatitis C virus 475 Ala Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala ArgThr Thr Gln Gly 1 5 10 15 Leu Val Ser Leu Phe Ser 20 476 24 PRTHepatitis C virus 476 Ala Arg Thr Thr Gln Gly Leu Val Ser Leu Phe SerArg Gly Ala Lys 1 5 10 15 Gln Asp Ile Gln Leu Ile Asn Thr 20 477 9 PRTHepatitis C virus 477 Ile Pro Lys Pro Gln Arg Lys Thr Lys 1 5 478 9 PRTHepatitis C virus 478 Pro Lys Pro Gln Arg Lys Thr Lys Arg 1 5 479 9 PRTHepatitis C virus 479 Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 480 9 PRTHepatitis C virus 480 Pro Gln Arg Lys Thr Lys Arg Asn Thr 1 5 481 9 PRTHepatitis C virus 481 Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 482 9 PRTHepatitis C virus 482 Arg Lys Thr Lys Arg Asn Thr Asn Arg 1 5 483 9 PRTHepatitis C virus 483 Lys Thr Lys Arg Asn Thr Asn Arg Arg 1 5 484 9 PRTHepatitis C virus 484 Thr Lys Arg Asn Thr Asn Arg Arg Pro 1 5 485 9 PRTHepatitis C virus 485 Arg Arg Pro Gln Asp Val Lys Phe Pro 1 5 486 9 PRTHepatitis C virus 486 Arg Pro Gln Asp Val Lys Phe Pro Gly 1 5 487 9 PRTHepatitis C virus 487 Pro Gln Asp Val Lys Phe Pro Gly Gly 1 5 488 9 PRTHepatitis C virus 488 Gln Asp Val Lys Phe Pro Gly Gly Gly 1 5 489 9 PRTHepatitis C virus 489 Asp Val Lys Phe Pro Gly Gly Gly Gln 1 5 490 9 PRTHepatitis C virus 490 Gly Gly Val Tyr Leu Leu Pro Arg Arg 1 5 491 9 PRTHepatitis C virus 491 Gly Val Tyr Leu Leu Pro Arg Arg Gly 1 5 492 9 PRTHepatitis C virus 492 Val Tyr Leu Leu Pro Arg Arg Gly Pro 1 5 493 9 PRTHepatitis C virus 493 Tyr Leu Leu Pro Arg Arg Gly Pro Arg 1 5 494 9 PRTHepatitis C virus 494 Leu Leu Pro Arg Arg Gly Pro Arg Leu 1 5 495 9 PRTHepatitis C virus 495 Leu Pro Arg Arg Gly Pro Arg Leu Gly 1 5 496 9 PRTHepatitis C virus 496 Pro Arg Arg Gly Pro Arg Leu Gly Val 1 5 497 9 PRTHepatitis C virus 497 Gly Pro Arg Leu Gly Val Arg Ala Thr 1 5 498 9 PRTHepatitis C virus 498 Pro Arg Leu Gly Val Arg Ala Thr Arg 1 5 499 9 PRTHepatitis C virus 499 Arg Leu Gly Val Arg Ala Thr Arg Lys 1 5 500 9 PRTHepatitis C virus 500 Glu Arg Ser Gln Pro Arg Gly Arg Arg 1 5 501 9 PRTHepatitis C virus 501 Arg Ser Gln Pro Arg Gly Arg Arg Gln 1 5 502 9 PRTHepatitis C virus 502 Ser Gln Pro Arg Gly Arg Arg Gln Pro 1 5 503 9 PRTHepatitis C virus 503 Arg Gly Arg Arg Gln Pro Ile Pro Lys 1 5 504 9 PRTHepatitis C virus 504 Gly Arg Arg Gln Pro Ile Pro Lys Val 1 5 505 9 PRTHepatitis C virus 505 Arg Arg Gln Pro Ile Pro Lys Val Arg 1 5 506 9 PRTHepatitis C virus 506 Pro Ile Pro Lys Val Arg Arg Pro Glu 1 5 507 9 PRTHepatitis C virus 507 Pro Glu Gly Arg Thr Trp Ala Gln Pro 1 5 508 9 PRTHepatitis C virus 508 Glu Gly Arg Thr Trp Ala Gln Pro Gly 1 5 509 9 PRTHepatitis C virus 509 Gly Arg Thr Trp Ala Gln Pro Gly Tyr 1 5 510 9 PRTHepatitis C virus 510 Arg Thr Trp Ala Gln Pro Gly Tyr Pro 1 5 511 9 PRTHepatitis C virus 511 Thr Trp Ala Gln Pro Gly Tyr Pro Trp 1 5 512 9 PRTHepatitis C virus 512 Trp Ala Gln Pro Gly Tyr Pro Trp Pro 1 5 513 9 PRTHepatitis C virus 513 Ala Gln Pro Gly Tyr Pro Trp Pro Leu 1 5 514 9 PRTHepatitis C virus 514 Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 1 5 515 9 PRTHepatitis C virus 515 Leu Ser Gly Lys Pro Ala Ile Ile Pro 1 5 516 9 PRTHepatitis C virus 516 Gly Lys Pro Ala Ile Ile Pro Asp Arg 1 5 517 9 PRTHepatitis C virus 517 Pro Ala Ile Ile Pro Asp Arg Glu Val 1 5 518 9 PRTHepatitis C virus 518 Ala Ile Ile Pro Asp Arg Glu Val Leu 1 5 519 9 PRTHepatitis C virus 519 Ile Ile Pro Asp Arg Glu Val Leu Tyr 1 5 520 9 PRTHepatitis C virus 520 Ile Pro Asp Arg Glu Val Leu Tyr Arg 1 5 521 9 PRTHepatitis C virus 521 Pro Asp Arg Glu Val Leu Tyr Arg Glu 1 5 522 9 PRTHepatitis C virus 522 Asp Arg Glu Val Leu Tyr Arg Glu Phe 1 5 523 9 PRTHepatitis C virus 523 Cys Ser Gln His Leu Pro Tyr Ile Glu 1 5 524 9 PRTHepatitis C virus 524 Ser Gln His Leu Pro Tyr Ile Glu Gln 1 5 525 9 PRTHepatitis C virus 525 Gln His Leu Pro Tyr Ile Glu Gln Gly 1 5 526 9 PRTHepatitis C virus 526 His Leu Pro Tyr Ile Glu Gln Gly Met 1 5 527 9 PRTHepatitis C virus 527 Leu Pro Tyr Ile Glu Gln Gly Met Met 1 5 528 9 PRTHepatitis C virus 528 Pro Tyr Ile Glu Gln Gly Met Met Leu 1 5 529 9 PRTHepatitis C virus 529 Tyr Ile Glu Gln Gly Met Met Leu Ala 1 5 530 9 PRTHepatitis C virus 530 Ile Glu Gln Gly Met Met Leu Ala Glu 1 5 531 9 PRTHepatitis C virus 531 Met Met Leu Ala Glu Gln Phe Lys Gln 1 5 532 9 PRTHepatitis C virus 532 Met Leu Ala Glu Gln Phe Lys Gln Lys 1 5 533 9 PRTHepatitis C virus 533 Leu Ala Glu Gln Phe Lys Gln Lys Ala 1 5 534 9 PRTHepatitis C virus 534 Ala Glu Gln Phe Lys Gln Lys Ala Leu 1 5 535 9 PRTHepatitis C virus 535 Glu Gln Phe Lys Gln Lys Ala Leu Gly 1 5 536 9 PRTHepatitis C virus 536 Gln Phe Lys Gln Lys Ala Leu Gly Leu 1 5 537 9 PRTHepatitis C virus 537 Phe Lys Gln Lys Ala Leu Gly Leu Leu 1 5 538 9 PRTHepatitis C virus 538 Lys Gln Lys Ala Leu Gly Leu Leu Gln 1 5 539 9 PRTHepatitis C virus 539 Gln Lys Ala Leu Gly Leu Leu Gln Thr 1 5 540 9 PRTHepatitis C virus 540 Lys Ala Leu Gly Leu Leu Gln Thr Ala 1 5 541 9 PRTHepatitis C virus 541 Ala Leu Gly Leu Leu Gln Thr Ala Ser 1 5 542 9 PRTHepatitis C virus 542 Leu Gly Leu Leu Gln Thr Ala Ser Arg 1 5 543 9 PRTHepatitis C virus 543 Gly Leu Leu Gln Thr Ala Ser Arg Gln 1 5 544 9 PRTHepatitis C virus 544 Leu Leu Gln Thr Ala Ser Arg Gln Ala 1 5 545 9 PRTHepatitis C virus 545 Ser Val Pro Ala Glu Ile Leu Arg Lys 1 5 546 9 PRTHepatitis C virus 546 Val Pro Ala Glu Ile Leu Arg Lys Ser 1 5 547 9 PRTHepatitis C virus 547 Pro Ala Glu Ile Leu Arg Lys Ser Arg 1 5 548 9 PRTHepatitis C virus 548 Ala Glu Ile Leu Arg Lys Ser Arg Arg 1 5 549 9 PRTHepatitis C virus 549 Glu Ile Leu Arg Lys Ser Arg Arg Phe 1 5 550 9 PRTHepatitis C virus 550 Phe Ala Gln Ala Leu Pro Val Trp Ala 1 5 551 9 PRTHepatitis C virus 551 Ala Gln Ala Leu Pro Val Trp Ala Arg 1 5 552 9 PRTHepatitis C virus 552 Gln Ala Leu Pro Val Trp Ala Arg Pro 1 5 553 9 PRTHepatitis C virus 553 Ala Leu Pro Val Trp Ala Arg Pro Asp 1 5 554 9 PRTHepatitis C virus 554 Val Trp Ala Arg Pro Asp Tyr Asn Pro 1 5 555 9 PRTHepatitis C virus 555 Trp Ala Arg Pro Asp Tyr Asn Pro Pro 1 5 556 9 PRTHepatitis C virus 556 Ala Arg Pro Asp Tyr Asn Pro Pro Leu 1 5 557 9 PRTHepatitis C virus 557 Arg Pro Asp Tyr Asn Pro Pro Leu Val 1 5 558 9 PRTHepatitis C virus 558 Pro Asp Tyr Asn Pro Pro Leu Val Glu 1 5 559 9 PRTHepatitis C virus 559 Pro Pro Leu Val Glu Thr Trp Lys Lys 1 5 560 9 PRTHepatitis C virus 560 Pro Leu Val Glu Thr Trp Lys Lys Pro 1 5 561 9 PRTHepatitis C virus 561 Leu Val Glu Thr Trp Lys Lys Pro Asp 1 5 562 9 PRTHepatitis C virus 562 Trp Glu Thr Trp Lys Lys Pro Asp Tyr 1 5 563 9 PRTHepatitis C virus 563 Glu Thr Trp Lys Lys Pro Asp Tyr Glu 1 5 564 9 PRTHepatitis C virus 564 Thr Trp Lys Lys Pro Asp Tyr Glu Pro 1 5 565 9 PRTHepatitis C virus 565 Trp Lys Lys Pro Asp Tyr Glu Pro Pro 1 5 566 9 PRTHepatitis C virus 566 Lys Lys Pro Asp Tyr Glu Pro Pro Val 1 5 567 9 PRTHepatitis C virus 567 Lys Pro Asp Tyr Glu Pro Pro Val Val 1 5 568 9 PRTHepatitis C virus 568 Pro Asp Tyr Glu Pro Pro Val Val His 1 5 569 9 PRTHepatitis C virus 569 Asp Tyr Glu Pro Pro Val Val His Gly 1 5 570 9 PRTHepatitis C virus 570 Tyr Glu Pro Pro Val Val His Gly Cys 1 5 571 9 PRTHepatitis C virus 571 Pro Pro Val Val His Gly Cys Pro Leu 1 5 572 9 PRTHepatitis C virus 572 Pro Val Val His Gly Cys Pro Leu Pro 1 5 573 9 PRTHepatitis C virus 573 Val Val His Gly Cys Pro Leu Pro Pro 1 5 574 9 PRTHepatitis C virus 574 Val His Gly Cys Pro Leu Pro Pro Lys 1 5 575 9 PRTHepatitis C virus 575 His Gly Cys Pro Leu Pro Pro Lys Ser 1 5 576 9 PRTHepatitis C virus 576 Ser Pro Pro Val Pro Pro Pro Arg Lys 1 5 577 6 PRTHepatitis C virus 577 Pro Gln Arg Lys Thr Lys 1 5 578 6 PRT Hepatitis Cvirus 578 Pro Gln Arg Lys Thr Lys 1 5 579 7 PRT Hepatitis C virus 579Pro Gln Asp Val Lys Phe Pro 1 5 580 6 PRT Hepatitis C virus 580 Tyr LeuLeu Pro Arg Arg 1 5 581 7 PRT Hepatitis C virus 581 Pro Arg Arg Gly ProArg Leu 1 5 582 7 PRT Hepatitis C virus 582 Arg Leu Gly Val Arg Ala Thr1 5 583 7 PRT Hepatitis C virus 583 Ser Gln Pro Arg Gly Arg Arg 1 5 5847 PRT Hepatitis C virus 584 Arg Arg Gln Pro Ile Pro Lys 1 5 585 6 PRTHepatitis C virus 585 Arg Thr Trp Ala Gln Pro 1 5 586 8 PRT Hepatitis Cvirus 586 Gln Pro Gly Tyr Pro Trp Pro Leu 1 5 587 6 PRT Hepatitis Cvirus 587 Pro Asp Arg Glu Val Leu 1 5 588 6 PRT Hepatitis C virus 588His Leu Pro Tyr Ile Glu 1 5 589 8 PRT Hepatitis C virus 589 Tyr Ile GluGln Gly Met Met Leu 1 5 590 7 PRT Hepatitis C virus 590 Ala Glu Gln PheLys Gln Lys 1 5 591 6 PRT Hepatitis C virus 591 Lys Gln Lys Ala Leu Gly1 5 592 7 PRT Hepatitis C virus 592 Leu Gly Leu Leu Gln Thr Ala 1 5 5937 PRT Hepatitis C virus 593 Pro Ala Glu Ile Leu Arg Lys 1 5 594 7 PRTHepatitis C virus 594 Glu Ile Leu Arg Lys Ser Arg 1 5 595 7 PRTHepatitis C virus 595 Gln Ala Leu Pro Val Trp Ala 1 5 596 6 PRTHepatitis C virus 596 Pro Asp Tyr Asn Pro Pro 1 5 597 7 PRT Hepatitis Cvirus 597 Leu Val Glu Thr Trp Lys Lys 1 5 598 6 PRT Hepatitis C virus598 Asp Tyr Glu Pro Pro Val 1 5 599 5 PRT Hepatitis C virus 599 His GlyCys Pro Leu 1 5 600 20 PRT Hepatitis C virus 600 Gly Arg Thr Trp Ala GlnPro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn 1 5 10 15 Glu Gly Cys Gly 20

What is claimed is:
 1. A peptide consisting of an amino acid sequence ofSEQ ID NO 454 (A)-GHTRVSGGAAASDTRGLVSLFSPGSAQKIQLVNT-(Z) (SEQ ID NO454), wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 454 which is immunologicallyreactive with HCV antisera.
 2. A peptide consisting of an amino acidsequence of SEQ ID NO: 454 (A)-GHTRVSGGAAASDTRGLVSLFSPGSAQKIQLVNT-(Z)(SEQ ID NO 454), wherein A, when present, represents an amino acid,amino group, or chemically modified amino terminus of the peptide, andwherein Z, when present, represents an amino acid, OH-group, NH₂-group,or a linkage involving an OH-group or an NH₂-group; or a peptidefragment consisting of at least 5 amino acids of SEQ ID NO: 454 which isimmunologically reactive with HCV antisera; and said peptide or peptidefragment containing at least one N-terminal biotin group, C-terminalbiotin group or biotin group attached to an internal amino acid; saidbiotin group being attached directly to the peptide or peptide fragmentor attached to the peptide or peptide fragment through a linker Y; saidlinker Y consisting of 1 to 10 chemical entities selected from the groupconsisting of a glycine residue, beta-alanine, 4-aminobutyric acid,5-aminovaleric acid and 6-aminohexanoic acid.
 3. A peptide complexcomprising the peptide or peptide fragment according to claim 2 coupledto at least one streptavidin molecule or avidin molecule.
 4. The peptidecomplex according to claim 3 wherein said streptavidin molecule oravidin molecule is coupled to a solid phase.
 5. A solid phase comprisinga peptide or peptide fragment according to claim 1 or 2 and a solidsupport wherein the solid support is anchored to the peptide via atleast one covalent or non-covalent bond.
 6. A solid phase according toclaim 5, wherein said solid support is a nylon membrane and said peptideor peptide fragment is anchored via a biotin group to streptavidinpresent on the nylon membrane.
 7. The solid phase according to claim 5wherein said solid support is a nylon membrane.
 8. An immunologicalassay kit for detecting antibodies to HCV comprising at least onepeptide or peptide fragment according to any of claim 1 or 2, or apeptide complex of claim
 3. 9. A Line immunoassay kit for detectingantibodies to HCV comprising at least one peptide or peptide fragmentaccording to any of claim 1 or 2, or a peptide complex of claim
 3. 10.An immunological assay kit for detecting antibodies to HCV present in abiological sample comprising at least one peptide or peptide fragmentaccording to any of claim 1 or 2, or a peptide complex of claim
 3. 11. ALine immunoassay kit for detecting antibodies to HCV present in abiological sample comprising at least one peptide or peptide fragmentaccording to any of claim 1 or 2, or a peptide complex of claim
 3. 12. Apeptide or peptide fragment of claim 1 or 2 wherein at least one of Aand Z are not present.
 13. A peptide complex of claim 3 wherein at leastone of A and Z are not present.
 14. The peptide complex according toclaim 13 wherein said streptavidin molecule or avidin molecule iscoupled to a solid phase.
 15. A sold phase comprising a peptide orpeptide fragment according to claim 12 and a solid support wherein thesolid support is anchored to the peptide via at least one covalent ornon-covalent bond.
 16. A solid phase according to claim 15, wherein saidsolid support is a nylon membrane and said peptide or peptide fragmentis anchored via a biotin group to streptavidin present on the nylonmembrane.
 17. The solid phase according to claim 15, wherein said solidsupport is a nylon membrane.
 18. An immunological assay kit fordetecting antibodies to HCV comprising at least one peptide or peptidefragment according to claim
 12. 19. A Line Immunoassay kit for detectingantibodies to HCV comprising at least one peptide or peptide fragmentaccording to claim
 12. 20. An immunological assay kit for detectingantibodies to HCV present in a biological sample comprising at least onepeptide or peptide fragment according to claim
 12. 21. A Lineimmunoassay kit for detecting antibodies to HCV present in a biologicalsample comprising at least one peptide or peptide fragment according toclaim
 12. 22. An immunological assay kit for detecting antibodies to HCVcomprising a peptide complex of claim
 13. 23. A Line immunoassay kit fordetecting antibodies to HCV comprising a peptide complex of claim 13.24. An immunological assay kit for detecting antibodies to HCV presentin a biological sample comprising a peptide complex of claim
 13. 25. ALine immunoassay kit for detecting antibodies to HCV present in abiological sample comprising a peptide complex of claim
 13. 26. Apeptide fragment according to claim 2, consisting of an amino acidsequence of SEQ ID NO 408; GHTRVSGGAAASDTRGLVSLFS (SEQ ID NO 408), whichis immunologically reactive with HCV antisera; and said peptide fragmentcontaining at least one N-terminal biotin group, C-terminal biotingroup, biotin group attached to an internal amino acid or no biotingroup; said biotin group, when present, being attached directly to thepeptide or peptide fragment or attached to the peptide or peptidefragment through a linker Y; said linker Y consisting of 1 to 10chemical entities selected from the group consisting of a glycineresidue, beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid and6-aminohexanoic acid.
 27. A peptide fragment according to claim 2consisting of an amino acid sequence of SEQ ID NO 409:ASDTRGLVSLFSPGSAQKIQLVNT (SEQ ID NO 409), which is immunologicallyreactive with HCV antisera; and said peptide fragment containing atleast one N-terminal biotin group, C-terminal biotin group, biotin groupattached to an internal amino acid or no biotin group; said biotingroup, which present, being attached directly to the peptide or peptidefragment or attached to the peptide or peptide fragment through a linkerY; said linker Y consisting of 1 to 10 chemical entities selected fromthe group consisting of a glycine residue! beta-alanine, 4-aminobutyricacid, 5-aminovaleric acid and 6-aminohexanoic acid.
 28. A peptidecomplex comprising a peptide fragment according to claim 26 or 27,coupled to at least one streptavidin molecule or avidin molecule. 29.The peptide complex according to claim 28, wherein said streptavidinmolecule or avidin molecule is coupled to a solid phase.
 30. A solidphase comprising a peptide fragment according to claim 26 or 27 and asolid support wherein the solid support is anchored to the peptidefragment via at least one covalent or non-covalent bond.
 31. A solidphase according to claim 30, wherein said solid support is a nylonmembrane and said peptide fragment is anchored via a biotin group tostreptavidin present on the nylon membrane.
 32. The solid phaseaccording to claim 30, wherein said solid support is a nylon membrane.33. An immunological assay kit for detecting antibodies to HCVcomprising at least one peptide fragment according to any of claim 26 or27.
 34. An immunological assay kit for detecting antibodies to HCVcomprising at least one peptide complex of claim
 28. 35. A Lineimmunoassay kit for detecting antibodies to HCV comprising at least onepeptide fragment according to any of claim 26 or
 27. 36. A Lineimmunoassay kit for detecting antibodies to HCV comprising at least onepeptide complex of claim
 28. 37. An immunological assay kit fordetecting antibodies to HCV present in a biological sample comprising atleast one peptide fragment according to any of claim 26 or
 27. 38. Animmunological assay kit for detecting antibodies to HCV present in abiological sample comprising at least one a peptide complex of claim 28.39. A Line immunoassay kit for detecting antibodies to HCV present in abiological sample comprising at least one peptide fragment according toany of claim 26 or
 27. 40. A Line immunoassay kit for detectingantibodies to HCV present in a biological sample comprising at least onepeptide complex of claim
 28. 41. A peptide consisting of an amino acidsequence of SEQ ID NO 454 (A)-THTRVSGGAAASDTRGLVSLFSPGSAQKIQLVNT-(Z)(SEQ ID NO 454), wherein A, when present, represents an amino acid,amino group, or chemically modified amino terminus of the peptide, andwherein Z, when present, represents an amino acid, OH-group, NH₂-group,or a linkage involving an OH-group or an NH₂-group; or a peptidefragment consisting of at least 5 amino acids of SEQ ID NO:
 454. 42. Apeptide consisting of an amino acid sequence of SEQ ID NO 454(A)-GHTRVSGGAAASDTRGLVSLFSPGSAQKIQLVNT-(Z) (SEQ ID NO 454), wherein A,when present, represents an amino acid, amino group, or chemicallymodified amino terminus of the peptide, and wherein Z, when present,represents an amino acid, OH-group, NH₂-group, or a linkage involving anOH-group or an NH₂-group; or a peptide fragment consisting of at least 5amino acids of SEQ ID NO: 454; and said peptide or peptide fragmentcontaining at least one N-terminal biotin group, C-terminal biotin groupor biotin group attached to an internal amino acid; said biotin groupbeing attached directly to the peptide or peptide fragment or attachedto the peptide or peptide fragment through a linker Y; said linker Yconsisting of 1 to 10 chemical entities selected from the groupconsisting of a glycine residue, beta-alanine, 4-aminobutyric acid,5-aminovaleric acid and 6-aminohexanoic acid.
 43. A peptide complexcomprising the peptide or peptide fragment according to claim 42 coupledto at least one streptavidin molecule or avidin molecule.
 44. Thepeptide complex according to claim 43 wherein said streptavidin moleculeor avidin molecule is coupled to a solid phase.
 45. A solid phasecomprising a peptide or peptide fragment according to claim 41 or 42 anda solid support wherein the solid support is anchored to the peptide viaat least one covalent or non-covalent bond.
 46. An immunological assaykit for detecting antibodies to HCV comprising at least one peptide orpeptide fragment according to any of claim 41 or 42, or a peptidecomplex or claim
 43. 47. A peptide or peptide fragment of claim 41 or 42wherein at least one of A and Z are not present.
 48. A peptide complexof claim 43 wherein at least one of A and Z are not present.
 49. Apeptide fragment according to claim 42 consisting of an amino acidsequence of SEQ ID NO 408: GHTRVSGGAAASDTRGLVSLFS (SEQ ID NO 408); andsaid peptide fragment containing at least one N-terminal biotin group,C-terminal biotin group, biotin group attached to an internal amino acidor no biotin group; said biotin group, when present, being attacheddirectly to the peptide or peptide fragment or attached to the peptideor peptide fragment through a linker Y; said linker Y consisting of 1 to10 chemical entities selected from the group consisting of a glycineresidue, beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid and6-aminohexanoic acid.
 50. A peptide fragment according to claim 42consisting of an amino acid sequence of SEQ ID NO 409:ASDTRGLVSLFSPGSAQKIQLVNT (SEQ ID NO 409); and said peptide fragmentcontaining at least one N-terminal biotin group, C-terminal biotingroup, biotin group attached to an internal amino acid or no biotingroup; said biotin group, when present, being attached directly to thepeptide or peptide fragment or attached to the peptide or peptidefragment through a linker Y; said linker Y consisting of 1 to 10chemical entities selected from the group consisting of a glycineresidue! beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid and6-aminohexanoic acid.
 51. A peptide complex comprising a peptidefragment according to claim 49 or 50 coupled to at least onestreptavidin molecule or avidin molecule.
 52. An immunological assay kitfor detecting antibodies to HCV comprising at least one peptide fragmentaccording to any of claim 49 or 50.